Sample records for factor 4e-binding protein

Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor4Ebindingproteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negat...

Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor4Ebindingproteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.

Biguanides, including metformin, buformin, and phenformin, are potential antitumorigenic agents and induce cell death during glucose deprivation, a cell condition that occurs in the tumor microenvironment. Here, we show that this selective killing of glucose-deprived cells is coupled with hyperactivation of eukaryotic initiation factor4E-bindingprotein 1 (4E-BP1), a negative regulator of translation initiation. We found, in fact, that the 4E-BP1 hyperactivation led to failure of the unfolded protein response (UPR), an endoplasmic reticulum-originated stress signaling pathway for cell survival. We also found that the 4E-BP1-mediated UPR inhibition occurred through a strong inhibition of the mTOR signaling pathway, a proven antitumor target. Importantly, the 4E-BP1 hyperactivation can be also seen in xenografted cancer cells through an in vivo biguanide treatment. Our findings indicate that antitumor action of biguanides can be mediated by 4E-BP1 hyperactivation, which results in UPR inhibition and selective cell killing when glucose is withdrawn.

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4Ebindingprotein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4Ebindingproteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

Full Text Available Eukaryotic mRNA degradation often initiates with the recruitment of the CCR4-NOT deadenylase complex and decay factors to the mRNA 3′ terminus. How the 3′-proximal decay machinery interacts with the 5′-terminal cap structure in order to engender mRNA decapping and 5′–3′ degradation is unclear. Human 4E-T is an eIF4E-bindingprotein that has been reported to promote mRNA decay, albeit via an unknown mechanism. Here, we show that 4E-T is a component of the mRNA decay machinery and interacts with factors including DDX6, LSM14, and the LSM1-7-PAT1 complex. We also provide evidence that 4E-T associates with, and enhances the decay of, mRNAs targeted by the CCR4-NOT deadenylase complex, including microRNA targets. Importantly, we demonstrate that 4E-T must interact with eIF4E to engender mRNA decay. Taken together, our data support a model where 4E-T promotes mRNA turnover by physically linking the 3′-terminal mRNA decay machinery to the 5′ cap via its interaction with eIF4E.

4E-BP1是真核细胞翻译起始因子4E(eIF4E)的特异性抑制物，近年研究显示，磷酸化形式的4E-BP1(P-4E-BP1)在多种恶性肿瘤如乳腺癌、宫颈癌、甲状腺、前列腺癌、大肠癌等中存在过表达，并且其表达与肿瘤的大小、侵袭、转移能力、预后等临床病理特征密切相关。因此，阐明P-4E-BP1的表达与肿瘤的生物学行为将有助于全面了解恶性肿瘤的发生、发展、侵袭及转移机制，同时对指导临床诊断和治疗，判断预后有重大意义。更重要的是，P-4E-BP1有可能成为治疗肿瘤的新靶点，为临床治疗肿瘤提供新思路。%4E-BP1 is the specific inhibitor of eukaryotic initiation factor 4E, recent studies have shown, phosphorylated 4E-bindingprotein 1(P-4E-BP1)overexpression has been observed in a variety of human cancers, including breast cancer, cervical cancer, thyroid cancer, prostate cancer, colorectal cancer, et al. Its expression level is associated with tumor size, invasion, metastasis, prognosis and other clinical and pathological features. Thus evaluate P-4E-BP1 expression level which is associated with tumor biological behavior will help us better understand the etiology, development, invasion and metastasis mechanisms of cancer.Moreover, P-4E-BP1 may become a new target for cancer treatment and provide new ideas for the clinical cancer treatment.

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.

Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4Ebinding an m²²⁷G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m⁷G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4Ebinds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m⁷G-cap. Nematode eIF4Ebinding to the m⁷G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.

The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m(7)G) or a trimethylguanosine (m(2,2,7)G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m(7)G and m(2,2,7)G caps. The eIF4E.m(7)GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the cap-binding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m(7)GpppG and m(2,2,7)GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m(7)G versus m(2,2,7)G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m(2,2,7)G cap.

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress. DOI: http://dx.doi.org/10.7554/eLife.21907.001 PMID:28177284

A two-hybrid screen with the translation initiation factor, eIF(iso)4E from Arabidopsis, identified a clone encoding a lipoxygenase type 2 [Freire, M.A., et al., 2000. Plant lipoxygenase 2 is a translation initiation factor-4E-bindingprotein. Plant Molecular Biology 44, 129-140], and three cDNA clones encoding the homologue of the mammalian BTF3 factor, the beta subunit of the nascent polypeptide-associated complex (NAC). Here we report on the interaction between the translation initiation factor eIF(iso)4E and AtBTF3. AtBTF3 protein is able to interact with the wheat initiation factors eIF4E and eIF(iso)4E. AtBTF3 contains a sequence related to the prototypic motif found on most of the 4E-bindingproteins, and competes with the translation initiation factor eIF(iso)4G for eIF4(iso)4Ebinding, in a two hybrid interference assay. These findings provide a molecular link between the translation initiation mechanism and the emergence of the nascent polypeptide chains.

Full Text Available Abstract Background Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus. Results Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-bindingprotein 4E-BP1, or the formation of the cap-binding complex eIF4F. Conclusions Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

Full Text Available West Nile virus (WNV is a (+ sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1 for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K and eukaryotic translation initiation factor4E-bindingprotein (4EBP pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor4E-bindingprotein/eukaryotic translation initiation factor4E-bindingprotein (4EBP/eIF4E interaction and eukaryotic initiation factor 4F (eIF4F complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6 and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

Proteins encoded by highly expressed genes evolve more slowly. This correlation is thought to arise owing to purifying selection against toxicity of misfolded proteins (that should be more crucial for highly expressed genes). It is now widely accepted that this individual (by-gene) effect is a dominant cause in protein evolution. Here, I show that in mammals, the evolutionary rate of a protein is much more strongly related to the evolutionary rate of coexpressed proteins (and proteins of the same biological pathway) than to the expression level of its encoding gene. The complexity of gene regulation (estimated by the numbers of transcription factor targets and regulatory microRNA targets in the encoding gene) is another important cause, which is much stronger than gene expression level. Proteins encoded by complexly regulated genes evolve more slowly. The intronic length and the ratio of intronic to coding sequence lengths also correlate negatively with protein evolutionary rate (which contradicts the expectation from the negative link between expression level and evolutionary rate). One more important factor, which is much stronger than gene expression level, is evolutionary age. More recent proteins evolve faster, and expression level of an encoding gene becomes quite a minor cause in the evolution of mammal proteins of metazoan origin. These data suggest that, in contrast to a widespread opinion, systemic factors dominate mammal protein evolution.

The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

Factor H related proteins comprise a group of five plasma proteins: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5, and each member of this group binds to the central complement component C3b. Mutations, genetic deletions, duplications or rearrangements in the individual CFHR genes are associated with a number of diseases including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathies (C3 glomerulonephritis (C3GN), dense deposit disease (DDD) and CFHR5 nephropathy), IgA nephropathy, age related macular degeneration (AMD) and systemic lupus erythematosus (SLE). Although complement regulatory functions were attributed to most of the members of the CFHR protein family, the precise role of each CFHR protein in complement activation and the exact contribution to disease pathology is still unclear. Recent publications show that CFHR proteins form homo- as well as heterodimers. Genetic abnormalities within the CFHR gene locus can result in hybrid proteins with affected dimerization or recognition domains which cause defective functions. Here we summarize the recent data about CFHR genes and proteins in order to better understand the role of CFHR proteins in complement activation and in complement associated diseases.

Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor4E-bindingprotein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor4E-bindingprotein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged with sarcopenia and the patients with disuse atrophy. PMID:27330287

Eukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E-eIF4G interaction is regulated by the eIF4Ebinding partners, eIF4E-bindingproteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.

DDX3 is a human RNA helicase with plethoric functions. Our previous studies have indicated that DDX3 is a transcriptional regulator and functions as a tumor suppressor. In this study, we use a bicistronic reporter to demonstrate that DDX3 specifically represses cap-dependent translation but enhances hepatitis C virus internal ribosome entry site-mediated translation in vivo in a helicase activity-independent manner. To elucidate how DDX3 modulates translation, we identified translation initiation factor eukaryotic initiation factor 4E (eIF4E) as a DDX3-binding partner. Interestingly, DDX3 utilizes a consensus eIF4E-binding sequence YIPPHLR to interact with the functionally important dorsal surface of eIF4E in a similar manner to other eIF4E-bindingproteins. Furthermore, cap affinity chromatography analysis suggests that DDX3 traps eIF4E in a translationally inactive complex by blocking interaction with eIF4G. Point mutations within the consensus eIF4E-binding motif in DDX3 impair its ability to bind eIF4E and result in a loss of DDX3's regulatory effects on translation. All these features together indicate that DDX3 is a new member of the eIF4E inhibitory proteins involved in translation initiation regulation. Most importantly, this DDX3-mediated translation regulation also confers the tumor suppressor function on DDX3. Altogether, this study demonstrates regulatory roles and action mechanisms for DDX3 in translation, cell growth and likely viral replication.

The human cytomegalovirus (HCMV) IE86 Cdna was cloned into Pgex-2T and fusion protein GST-IE86 was expressed in E. Coli. SDS-PAGE and Western blot assay indicated that fusion protein GST-IE86 with molecular weight of 92 ku is soluble in the supernatant of cell lysate. Protein GST and fusion protein GST-IE86 were purified by affinity chromatography. The technology of co-separation and specific affinity chromatography was used to study the interactions of HCMV IE86 protein with some transcriptional regulatory proteins and transcriptional factors. The results indicated that IE86 interacts separately with transcriptional factor TFIIB and promoter DNA binding transcription trans-activating factors SP1, AP1 and AP2 to form a heterogenous protein complex. These transcriptional trans-activating factors, transcriptional factor and IE86 protein were adsorbed and retained in the affinity chromatography simultaneously. But IE86 protein could not interact with NF-Кb, suggesting that the function of IE86 protein that can interact with transcriptional factor and transcriptional trans-activating factors has no relevance to protein glycosylation. IE86 protein probably has two domains responsible for binding transcriptional trans-activating regulatory proteins and transcriptional factors respectively, thus activating the transcription of many genes. The interactions accelerated the assembly of the transcriptional initiation complexes.

Protein content of any source is classically determined through the analysis of its nitrogen content done for more 100 years by the Kjeldahl method, and the obtained result is multiplied by a number named nitrogen conversion factor (NCF). The value of NCF is related to the amino acid composition of the protein source and to the eventual presence of side groups covalently bound to some amino acids of the protein chain. Consequently, the value of NCF cannot be identical for all sources of food proteins. The aim of this paper is to review the available knowledge on the two allowed protein sources for infant food formulas, milk and soybean, in order to bring the right scientific basis which should be used for the revision of both European legislation and Codex Standard for Infant Formulas.

It has been almost 20 years since the first report of a WRKY transcription factor,SPF1,from sweet potato.Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth,development,and responses to biotic and abiotic stress.Despite the functional diversity,almost all analyzed WRKY proteins recognize the TrGACC/T W-box sequences and,therefore,mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors.Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling,transcription,and chromatin remodeling.Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors.It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes.In this review,we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute,at different levels,to the establishment of the complex regulatory and functional network of WRKY transcription factors.

We use a three-dimensional lattice model of proteins to investigate systematically the global properties of the polypeptide chains that determine the folding to the native conformation starting from an ensemble of denatured conformations. In the coarse-grained description, the polypeptide chain is modeled as a heteropolymer consisting of N beads confined to the vertices of a simple cubic lattice. The interactions between the beads are taken from a random gaussian distribution of energies, with a mean value B0 Monte Carlo simulations. The kinetics of approach to the native state for all the sequences was obtained using Monte Carlo simulations. For all sequences we find that there are two intrinsic characteristic temperatures, namely, T theta and Tf. At the temperature T theta the polypeptide chain makes a transition to a collapsed structure, while at Tf the chain undergoes a transition to the native conformation. We show that foldability of sequences can be characterized entirely in terms of these two temperatures. It is shown that fast folding sequences have small values of sigma = (T theta - Tf)/T theta whereas slow folders have larger values of sigma (the range of sigma is 0 sigma sigma. An increase in sigma from 0.1 to 0.7 can result in an increase of 5-6 orders of magnitudes in folding times. In contrast, we demonstrate that there is no useful correlation between folding times and the energy gap between the native conformation and the first excited state at any N for any value of B0. In particular, in the parameter space of the model, many sequences with varying energy gaps, all with roughly the same folding time, can be easily engineered. Folding sequences in this model, therefore, can be classified based solely on the value of sigma. Fast folders have small values of sigma (typically less than about 0.1), moderate folders have values of sigma in the approximate range between 0.1 and 0.6, while for slow folders sigma exceeds 0.6. The precise boundary between

The effects of some dietary factors, other than source and amount of N and carbohydrate, on the amount and efficiency of microbial protein synthesis are discussed in this review. Specifically, these factors include dry matter intake of animals, forage:concentrate ratio of diets, rate of N and carbohydrate degradation, synchronized release of N and energy from diets, rate of passage, and other factors, such as vitamins and minerals. It seemed that diets containing a mixture of forages and conc...

Full Text Available Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and

Obesity has reached epidemic proportions worldwide. The health consequences of obesity are more dangerous when associated with the metabolic syndrome and its components. Studies show that whey protein and its bioactive components can promote greater benefits compared to other protein sources such as egg and casein. The aim of this paper is to review the effects of whey protein on cardiometabolic risk factors. Using PubMed as the database, a review was conducted to identify current scientific literature on whey protein and the components of the metabolic syndrome published between 1970 and 2012. Consumption of whey protein seems to play an anti-obesity and muscle-protective role during dieting by increasing thermogenesis and maintaining lean mass. In addition, whey protein has been shown to improve glucose levels and insulin response, promote a reduction in blood pressure and arterial stiffness, and improve lipid profile. The collective view of current scientific literature indicates that the consumption of whey protein may have beneficial effects on some symptoms of the metabolic syndrome as well as a reduction in cardiovascular risk factors.

Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

Full Text Available Abstract Background Hox proteins are transcription factors involved in crucial processes during animal development. Their mode of action remains scantily documented. While other families of transcription factors, like Smad or Stat, are known cell signaling transducers, such a function has never been squarely addressed for Hox proteins. Results To investigate the mode of action of mammalian Hoxa1, we characterized its interactome by a systematic yeast two-hybrid screening against ~12,200 ORF-derived polypeptides. Fifty nine interactors were identified of which 45 could be confirmed by affinity co-purification in animal cell lines. Many Hoxa1 interactors are proteins involved in cell-signaling transduction, cell adhesion and vesicular trafficking. Forty-one interactions were detectable in live cells by Bimolecular Fluorescence Complementation which revealed distinctive intracellular patterns for these interactions consistent with the selective recruitment of Hoxa1 by subgroups of partner proteins at vesicular, cytoplasmic or nuclear compartments. Conclusions The characterization of the Hoxa1 interactome presented here suggests unexplored roles for Hox proteins in cell-to-cell communication and cell physiology.

Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription.

ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-β receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

Full Text Available BACKGROUND: Transcription factors of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1 family are key regulators of metazoan development and function as the effector components of the Notch receptor signalling pathway implicated in various cell fate decisions. CSL proteins recognize specifically the GTG[G/A]AA sequence motif and several mutants compromised in their ability to bind DNA have been reported. In our previous studies we have identified a number of novel putative CSL family members in fungi, organisms lacking the Notch pathway. It is not clear whether these represent genuine CSL family members. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of in vitro and in vivo approaches we characterized the DNA binding properties of Cbf11 and Cbf12, the antagonistic CSL paralogs from the fission yeast, important for the proper coordination of cell cycle events and the regulation of cell adhesion. We have shown that a mutation of a conserved arginine residue abolishes DNA binding in both CSL paralogs, similar to the situation in mouse. We have also demonstrated the ability of Cbf11 and Cbf12 to activate gene expression in an autologous fission yeast reporter system. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the fission yeast CSL proteins are indeed genuine family members capable of functioning as transcription factors, and provide support for the ancient evolutionary origin of this important protein family.

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale.

Leptin was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it may work as an autocrine hormone, mediating angiogenesis, growth, and immunomodulation. Leptin receptor (LEPR, also known as Ob-R) shows sequence homology to members of the class I cytokine receptor (gp130) superfamily. In fact, leptin may function as a proinflammatory cytokine. We have previously found that leptin is a trophic and mitogenic factor for trophoblastic cells. In order to further investigate the mechanism by which leptin stimulates cell growth in JEG-3 cells and trophoblastic cells, we studied the phosphorylation state of different proteins of the initiation stage of translation and the total protein synthesis by [(3)H]leucine incorporation in JEG-3 cells. We have found that leptin dose-dependently stimulates the phosphorylation and activation of the translation initiation factor EIF4E as well as the phosphorylation of the EIF4Ebindingprotein EIF4EBP1 (PHAS-I), which releases EIF4E to form active complexes. Moreover, leptin dose-dependently stimulates protein synthesis, and this effect can be partially prevented by blocking mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PIK3) pathways. In conclusion, leptin stimulates protein synthesis, at least in part activating the translation machinery, via the activation of MAPK and PIK3 pathways.

Full Text Available Proteins of the same functional family (for example, kinases may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content tend to have longer genes than species with low GC3 content.

Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcriptionally regulated in alveolar epithelial cells by hypoxia inducible factor (HIF). Previous findings in a newborn mouse model of bronchopulmonary dysplasia (BPD) suggest that thioredoxin interacting protein (Txnip) is a novel regulator of VEGF expression. The present studies were designed to test the hypothesis that Txnip negatively regulates VEGF through effects on HIF-mediated gene expression. To test this hypothesis, we first examined the levels of VEGF and Txnip protein in the lungs of 1 day-old newborn and E19 embryos and detected a significant inverse correlation. To elucidate the mechanisms underlying this relationship, we studied the effects of Txnip overexpression on HIF-mediated transcription using murine lung epithelial (MLE-12) cells. Overexpression of Txnip inhibited HIF-mediated reporter activity in both hypoxia and room air. Suppression of HIF activity by Txnip appeared to be independent of the ability of Txnip to bind to thioredoxin. Thus, our studies support a model in which Txnip is a potentially critical regulator of HIF-mediated gene transcription in the murine lung. Alterations in Txnip expression could alter lung VEGF expression in prematurely born human infants and contribute to the development of BPD. PMID:20692333

The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to constructprotein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and proteinprotein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale.

Anticoagulants are pivotal for the treatment of debilitating thromboembolic and associated disorders. Current anticoagulants such as heparin and warfarin are non-specific and have a narrow therapeutic window. These limitations have provided the impetus to develop new anticoagulant therapies/strategies that target specific factors in the blood coagulation cascade, ideally those located upstream in the clotting process. Factor VIIa (FVIIa) presents an attractive target as it, in complex with tissue factor (TF), acts as the prima ballerina for the formation of blood clot. A comprehensive review delineating the structure-activity relationship of protein/peptide anticoagulants targeting FVIIa or TF-FVIIa complex is absent in the literature. In this article, we have addressed this deficit by appraising the peptide/protein anticoagulants that target FVIIa/TF-FVIIa complex. Further, the current status of these anticoagulants, with regard to their performance in different clinical trials has also been presented. Lastly, the unexplored domains of these unique proteins have also been highlighted, which will facilitate further translational research in this paradigm, to improve strategies to counter and treat thromboembolic disorders.

Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif-containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens.

In biologic fluids, insulinlike growth factors (IGF-I and IGF-II) are bound to high-affinity insulinlike growth factor binding proteins (IGFBPs) of which seven have now been identified (IGFBPs 1-7). In a variety of biologic fluids, several IGFBPs undergo proteolytic degradation. Such degradation can lead to increased IGF bioavailability at the cell surface, facilitating receptor interactions. Herein, recent data identifying several IGFBP-degrading proteinases and their effects on IGF bioactivity is reviewed, and how IGFBP proteolysis is regulated by IGFs and IGFBPs, as well as how IGFBP cleavage analysis provides insights into the structure and function of IGFBPs, is explored. (Trends Endocrinol Metab 1997;8:299-306). (c) 1997, Elsevier Science Inc.

Full Text Available Transforming growth factor beta (TGF-β proteins are multifunctional cytokines whose neural functions are increasingly recognized. The machinery of TGF-β signaling, including the serine kinase type transmembrane receptors, is present in the central nervous system. However, the 3 mammalian TGF-β subtypes have distinct distributions in the brain suggesting different neural functions. Evidence of their involvement in the development and plasticity of the nervous system as well as their functions in peripheral organs suggested that they also exhibit neuroprotective functions. Indeed, TGF-β expression is induced following a variety of types of brain tissue injury. The neuroprotective function of TGF-βs is most established following brain ischemia. Damage in experimental animal models of global and focal ischemia was shown to be attenuated by TGF-βs. In addition, support for their neuroprotective actions following trauma, sclerosis multiplex, neurodegenerative diseases, infections, and brain tumors is also accumulating. The review will also describe the potential mechanisms of neuroprotection exerted by TGF-βs including anti-inflammatory, -apoptotic, -excitotoxic actions as well as the promotion of scar formation, angiogenesis, and neuroregeneration. The participation of these mechanisms in the neuroprotective effects of TGF-βs during different brain lesions will also be discussed.

It is now well known that the primary interactions of biological entities (e. g., tissues and cells) with nano-particles (NPs) are strongly influenced by the protein composition of the "corona" (i. e., the NP surface attached proteins). The composition of the corona strongly depends on the protein s

High growth rates in modern-day broilers require diets concentrated in digestible protein and energy. In addition to affecting feed conversion efficiency, it is important to prevent surplus dietary protein because of greater amounts of undigested protein entering the hindgut that may be fermented by

It is well known that ingestion of a protein source is effective in stimulating muscle protein synthesis after exercise. In addition, there are numerous reports on the impact of leucine and leucine-rich whey protein on muscle protein synthesis and mammalian target of rapamycin (mTOR) signalling. However, there is only limited information on the effects of whey protein hydrolysates (WPH) on muscle protein synthesis and mTOR signalling. The aim of the present study was to compare the effects of WPH and amino acids on muscle protein synthesis and the initiation of translation in skeletal muscle during the post-exercise phase. Male Sprague–Dawley rats swam for 2 h to depress muscle protein synthesis. Immediately after exercise, the animals were administered either carbohydrate (CHO), CHO plus an amino acid mixture (AA) or CHO plus WPH. At 1 h after exercise, the supplements containing whey-based protein (AA and WPH) caused a significant increase in the fractional rate of protein synthesis (FSR) compared with CHO. WPH also caused a significant increase in FSR compared with AA. Post-exercise ingestion of WPH caused a significant increase in the phosphorylation of mTOR levels compared with AA or CHO. In addition, WPH caused greater phosphorylation of ribosomal protein S6 kinase and eukaryotic initiation factor4E-bindingprotein 1 than AA and CHO. In contrast, there was no difference in plasma amino acid levels following supplementation with either AA or WPH. These results indicate that WPH may include active components that are superior to amino acids for stimulating muscle protein synthesis and initiating translation.

Use of recombinant human proteins has revolutionized medicine by providing over 200 highly purified hormones and proteins that effectively treat many inherited and acquired peptide hormone and protein deficiencies. With the exception of therapeutic monoclonal antibodies, these biological medicines are synthesized by cultured cells using DNA sequences that would yield proteins with identical amino acid sequences as endogenous human proteins. Therefore, there was the broad expectation that recombinant human biological medicines would be non-immunogenic in patients capable of synthesizing even sub-optimal levels of these therapeutic proteins to which they are innately tolerant. However, the widespread clinical use of recombinant human proteins has demonstrated that nearly all of them are immunogenic. This observation suggests that factors additional to differences in amino acid sequences of endogenous and biotherapeutic proteins contribute to the immunogenicity of therapeutic proteins. The main aim of this review is to summarize some of the factors that are known to contribute to the immunogenicity of recombinant therapeutic proteins.

Alzheimer’s, Huntington’s and Parkinson’s disease belong to a group of disorders caused by misfolded proteins that are known to stick together in a specific manner forming long fibrils made up of thousands of individual proteins. These fibrils are known as amyloids. It is still unclear which part of

Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.

Growth factor-induced proliferation and differentiation often require adhesion of cells to the extracellular matrix proteins such as fibronectin(FN).In this study,we aimed to investigate the effect of protein engineering of the keratinocyte growth factor 2(KGF2)fused to the FN on the mitogenic activity of KGF2.The fusion protein(KGF2-FN10),which was expressed in Escherichia coli,showed significantly enhanced mitogenic activity of KGF2 on human keratinocytes.Moreover,KGF2-FN10 fusion protein showed significantly increased activity to differentiate keratinocytes from native KGF2.In conclusion,these results suggest that KGF2-FN10 fusion protein has certain advantages over native KGF2 and may offer a novel strategy to potentiate the therapeutic effect of KGF2.

Digestibility of protein in traditional diets from developing countries such as India, Guatemala, and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94%). The presence of less digestible protein fractions, high levels of insoluble fiber, and high concentrations of antinutritional factors in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, are responsible for poor digestibility of protein. The effects of the presence of some of the important antinutritional factors on protein and amino digestibilities of food and feed products are reviewed in this chapter. Food and feed products may contain a number of antinutritional factors that may adversely affect protein digestibility and amino acid availability. Antinutritional factors may occur naturally, such as glucosinolates in mustard and rapeseed protein products, trypsin inhibitors and hemagglutinins in legumes, tannins in legumes and cereals, phytates in cereals and oilseeds, and gossypol in cottonseed protein products. Antinutritional factors may also be formed during heat/alkaline processing of protein products, yielding Maillard compounds, oxidized forms of sulfur amino acids, D-amino acids, and lysinoalanine (LAL, an unnatural amino acid derivative). The presence of high levels of dietary trypsin inhibitors from soybeans, kidney beans, or other grain legumes can cause substantial reductions in protein and amino acid digestibilities (up to 50%) in rats and pigs. Similarly, the presence of high levels of tannins in cereals, such as sorghum, and grain legumes, such as fababean (Vicia faba L.), can result in significantly reduced protein and amino acid digestibilities (up to 23%) in rats, poultry, and pigs. Studies involving phytase supplementation of production rations for swine or poultry have provided indirect evidence that normally encountered levels of phytates in cereals and legumes

Sep 1, 2000 ... Request for reprints to: Professor J. K. Tumwine, Department of Paediatrics and Child Health, ... severe forms of protein energy malnutrition among children ... were administered to the mothers or caretakers by one of us.

Protein–protein interactions are intrinsic to virtually every cellular process. Recent breakthroughs in techniques to study protein-interaction and the availability of fully sequenced plant genomes have attracted many plant scientists to undertake the first steps in the field of protein

Protein–protein interactions are intrinsic to virtually every cellular process. Recent breakthroughs in techniques to study protein-interaction and the availability of fully sequenced plant genomes have attracted many plant scientists to undertake the first steps in the field of protein interactions

The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The inducing material virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding {sup 3}H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein.

The lactating mammary gland is a prodigious protein-producing factory, but the milk protein synthesis mechanisms are not well understood. The major objective of this paper was to elucidate which genes and pathways were involved in the regulation of milk protein synthesis in the dairy goat mammary gland. Total 36 primiparous Guanzhong dairy goats were allotted in 12 groups according to their mammary development stages: days 90 and 150 of virgin, days 30, 90, and 150 of pregnancy, days 1, 10, 35, and 60 of lactation and days 3, 7, and 21 of involution (three animals per group). Mammary tissue RNA was isolated for quantitative real-time RT-PCR of four casein genes alpha-s1 casein (CSN1S1), alpha-s2 casein (CSN1S2), beta-casein (CSN2) and casein kappa (CSN3), four whey protein genes lactoglobulin (LGB), lactalbumin (LALBA), lactofarrin (LTF), and Whey acidic protein (WAP) and the genes which were potentially to regulate dairy goat milk protein synthesis at the level of transcription or translation [prolactin receptor (PRLR), AKT1, signal transducers and activators of transcription 5 (STAT5), E74-Like Factor 5 (ELF5), eukaryotic translation initiation factor4Ebindingprotein 1 (EIF4E-BP1), S6kinase (S6K) and caveolin 1]. The results showed that all genes were up-regulated in lactation period. The expressions of PRLR, AKT1, STAT5, ELF5, and S6K were similar to mRNA expressions of milk proteins. Our results indicated that milk protein synthesis in dairy goat mammary gland was possibly regulated by these genes.

To optimize the extraction of sorghum proteins, several variables were examined: sample-to-solvent ratio, detergent type and concentration, reducing agent type and concentration, extraction time, and buffer pH and concentration. Samples were quantified and characterized by RP-HPLC, FZCE, and nitrogen analysis. These studies revealed that pH, detergent type, reducing agent type, and sample-to-solvent ratio all had significant effects on the levels of protein extracted. Increasing SDS concentration (2%) and solvent-to-flour ratio (20:1) with multiple 5 min extracts reduced extraction time by 35-80% while still extracting the same levels of total protein relative to the control methodology. Reproducibility using the multiple extractions was found to be excellent with relative standard deviations of extractions.

Protein pharmaceuticals have matured into an important class of drugs, now comprising one in three novel drugs introduced on the market. However, significant gains are still to be made in reducing the costs of production, ensuring proper pharmacokinetics and efficacy, increasing patient compliance...

The delivery of low-density lipoprotein-derived cholesterol (LDL-C) from endosomal compartments to the plasma membrane and the endoplasmic reticulum (ER) is an important yet poorly understood cellular process.NiemannPick C1 (NPC1),a multi-pass integral membrane protein on the limiting membranes of late endosomes (LE)/lysosomes (Ly),is known to insert lumenal LDL-C to the limiting membrane of LE/Ly.Recent progress has identified novel cytoplasmic proteins that regulate the exit of LDL-C from LE/Ly,such as ORP5,a member of the oxysterolbinding protein-related protein (ORPs) family,and Hrs/VPS27,a well-established regulator of the endosomal sorting complex required for transport pathway.Whereas ORP5/ORPs may serve as cytosolic cholesterol carriers and deliver cholesterol in a non-vesicular manner,how Hrs/VPS27 regulate endosomal cholesterol sorting remains enigmatic.We discuss the functional relationship between NPC1,Hrs,and ORP5,and formulate possible schemes on how LDL-C may be moved from endosomal compartments to other cellular organelles.

Protein oxidation, the process caused especially by reactive oxygen and nitrogen species, is thought to play a major role in various oxidative processes within cells and is implicated in the development of many human diseases. This review provides a brief overview of the protein oxidation with the emphasis on the types of oxidation (oxidation of protein backbone and amino acid residues side chains, site-specific metal-catalysed protein oxidation), oxidation-dependent generation of protein hydroperoxides, carbonyl derivatives and protein–protein cross-linkages. Non-enzymatic glycoxidation (also known as Maillard reaction) as an important factor of protein damage, consequences of oxidative protein impairment and related diseases as well as means of monitoring and assessment of protein modifications are discussed.

α-Ketoglutarate (AKG) is a key intermediate in glutamine metabolism. Emerging evidence shows beneficial effects of AKG on clinical and experimental nutrition, particularly with respect to intestinal growth and integrity. However, the underlying mechanisms are unknown. Intestinal porcine epithelial cells (IPEC-1) were used to test the hypothesis that AKG inhibits glutamine degradation and enhances protein synthesis. IPEC-1 cells were cultured for 3 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 0, 0.2, 0.5 or 2 mM of AKG. At the end of the 3-day culture, cells were used to determine L-[U-14C]glutamine utilization, protein concentration, protein synthesis, and the total and phosphorylated levels of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-bindingprotein-1 (4E-BP1). Compared with 0 mM of AKG (control), 0.2 and 0.5 mM of AKG dose-dependently reduced (P<0.05) glutamine degradation and the production of glutamate, alanine and aspartate in IPEC-1 cells. Addition of 0.5 and 2 mM of AKG to culture medium enhanced protein synthesis (P<0.05) by 78 and 101% without affecting protein degradation, compared to the control group. Rapamycin (50 nM; a potent inhibitor of mTOR) attenuated the stimulatory effect of AKG on protein synthesis. Consistent with these metabolic data, the addition of 0.5 or 2 mM of AKG to culture medium increased (P<0.05) the phosphorylated levels of mTOR, S6k1 and 4E-BP1 proteins. Collectively, these results indicate that AKG can spare glutamine and activate the mTOR signaling pathway to stimulate protein synthesis in intestinal epithelial cells.

Solubility of Simplesse 100, the only whey-based fat substitute, was found to be good, considering the fact that technology for preparation of Simplesse 100 is a sequence of thermal steps. To characterize this phenomen, gel chromatography on Sephadex G-100, Sephacryl S-1000 and SDS-PAGE were used, supported by high-speed separation, UV studies and analytical procedures. Results show that the unusual solubility characteristic of microparticulated whey protein is related to two molecular effects: (1) optimal defolding of protein molecules and (2) stabilization of the defolded status by carbohydrate. Both effects were considered to favour non-covalent bonds, which contribute to the outstanding physico-functional and nutritive properties of microparticles.

Full Text Available Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ and the Nuclear factor κB (NF-κB component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis.

As a transcription factor, Yin Yang 1 (YY1) regulates the transcription of a dazzling list of genes and the number of its targets still mounts. Recent studies revealed that YY1 possesses functions independent of its DNA binding activity and its regulatory role in tumorigenesis has started to emerge.

Background:Initiation of transcription is essential for most of the cellular responses to environmental conditions and for cell and tissue specificity. This process is regulated through numerous proteins, their ligands and mutual interactions, as well as interactions with DNA. The key such regulatory proteins are transcription factors (TFs) and transcription co-factors (TcoFs). TcoFs are important since they modulate the transcription initiation process through interaction with TFs. In eukaryotes, transcription requires that TFs form different protein complexes with various nuclear proteins. To better understand transcription regulation, it is important to know the functional class of proteins interacting with TFs during transcription initiation. Such information is not fully available, since not all proteins that act as TFs or TcoFs are yet annotated as such, due to generally partial functional annotation of proteins. In this study we have developed a method to predict, using only sequence composition of the interacting proteins, the functional class of human TF binding partners to be (i) TF, (ii) TcoF, or (iii) other nuclear protein. This allows for complementing the annotation of the currently known pool of nuclear proteins. Since only the knowledge of protein sequences is required in addition to protein interaction, the method should be easily applicable to many species.Results:Based on experimentally validated interactions between human TFs with different TFs, TcoFs and other nuclear proteins, our two classification systems (implemented as a web-based application) achieve high accuracies in distinguishing TFs and TcoFs from other nuclear proteins, and TFs from TcoFs respectively.Conclusion:As demonstrated, given the fact that two proteins are capable of forming direct physical interactions and using only information about their sequence composition, we have developed a completely new method for predicting a functional class of TF interacting protein partners

The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter-residue interactions, ion-pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic-mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three-dimensional structures of elongation factor-Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the

Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.

Full Text Available Helicobacter pylori is a Gram-negative bacteria that is responsible for gastritis in human. Its spiral flagellated body helps in locomotion and colonization in the host environment. It is capable of living in the highly acidic environment of the stomach with the help of acid adaptive genes. The genome of H. pylori 26695 strain contains 1,555 coding genes that encode 1,445 proteins. Out of these, 340 proteins are characterized as hypothetical proteins (HP. This study involves extensive analysis of the HPs using an established pipeline which comprises various bioinformatics tools and databases to find out probable functions of the HPs and identification of virulence factors. After extensive analysis of all the 340 HPs, we found that 104 HPs are showing characteristic similarities with the proteins with known functions. Thus, on the basis of such similarities, we assigned probable functions to 104 HPs with high confidence and precision. All the predicted HPs contain representative members of diverse functional classes of proteins such as enzymes, transporters, binding proteins, regulatory proteins, proteins involved in cellular processes and other proteins with miscellaneous functions. Therefore, we classified 104 HPs into aforementioned functional groups. During the virulence factors analysis of the HPs, we found 11 HPs are showing significant virulence. The identification of virulence proteins with the help their predicted functions may pave the way for drug target estimation and development of effective drug to counter the activity of that protein.

Helicobacter pylori is a Gram-negative bacteria that is responsible for gastritis in human. Its spiral flagellated body helps in locomotion and colonization in the host environment. It is capable of living in the highly acidic environment of the stomach with the help of acid adaptive genes. The genome of H. pylori 26695 strain contains 1,555 coding genes that encode 1,445 proteins. Out of these, 340 proteins are characterized as hypothetical proteins (HP). This study involves extensive analysis of the HPs using an established pipeline which comprises various bioinformatics tools and databases to find out probable functions of the HPs and identification of virulence factors. After extensive analysis of all the 340 HPs, we found that 104 HPs are showing characteristic similarities with the proteins with known functions. Thus, on the basis of such similarities, we assigned probable functions to 104 HPs with high confidence and precision. All the predicted HPs contain representative members of diverse functional classes of proteins such as enzymes, transporters, binding proteins, regulatory proteins, proteins involved in cellular processes and other proteins with miscellaneous functions. Therefore, we classified 104 HPs into aforementioned functional groups. During the virulence factors analysis of the HPs, we found 11 HPs are showing significant virulence. The identification of virulence proteins with the help their predicted functions may pave the way for drug target estimation and development of effective drug to counter the activity of that protein. PMID:27729842

Recently, several proteins with homology to angiopoietins have been discovered. Three members of this new group, designated angiopoietin-like proteins (ANGPTLs), have been linked to regulation of energy metabolism. This review will focus on the fasting-induced adipose factor (FIAF)/ANGPTL4 as an

Full Text Available Hepatitis C virus (HCV enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1, which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion.

Steadily increasing evidence supports the idea that genetic diversities in the vascular bed are, in addition to hemodynamic influences, a major contributing factor in determining region-specific cardiovascular disease susceptibility. Members of the phylogenetically highly conserved Hox gene family of developmental regulators have to be viewed as prime candidates for determining these regional genetic differences in the vasculature. During embryonic patterning, the regionally distinct and precisely choreographed expression patterns of HOX transcription factors are essential for the correct specification of positional identities. Apparently, these topographic patterns are to some degree retained in certain adult tissues, including the circulatory system. While an understanding of the functional significance of these localized Hox activities in adult blood vessels is only beginning to emerge, an argument can be made for a role of Hox genes in the maintenance of vessel wall homeostasis and functional integrity on the one hand, and in regulating the development and progression of regionally restricted vascular pathologies, on the other. Initial functional studies in animal models, as well as data from clinical studies provide some level of support for this view. The data suggest that putative genetic regulatory networks of Hox-dependent cardiovascular disease processes include genes of diverse functional categories(extracellular matrix remodeling, transmembrane signaling, cell cycle control, inflammatory response, transcriptional control, etc.), as potential targets in both vascular smooth muscle and endothelial cells, as well as cell populations residing in the adventitia.

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

Francisella tularensis is a highly virulent intracellular pathogen with the capacity to infect a variety of hosts including humans. One of the most important proteins involved in F. tularensis virulence and pathogenesis is the protein DsbA. This protein is annotated as a lipoprotein with disulfide oxidoreductase/isomerase activity. Therefore, its interactions with different substrates, including probable virulence factors, to assist in their proper folding are anticipated. We aimed to use the immunopurification approach to find DsbA (gene locus FTS_1067) interacting partners in F. tularensis subsp. holarctica strain FSC200 and compare the identified substrates with proteins which were found in our previous comparative proteome analysis. As a result of our work two FTS_1067 substrates, D-alanyl-D-alanine carboxypeptidase family protein and HlyD family secretion protein, were identified. Bacterial two-hybrid systems were further used to test their relevance in confirming FTS_1067 protein interactions.

Full Text Available Hematopoietic stem cells (HSCs originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5 enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis.

Silencing Amblyomma americanum insulin-like growth factor binding protein-related protein 1 (AamIGFBP-rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP-rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP-1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP-rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24-48 h after attachment. Our data suggest that native AamIGFBP-rP1 is a functional insulin binding protein in that both yeast- and insect cell-expressed rAamIGFBP-rP1 bound insulin, but not insulin-like growth factors. When subjected to anti-blood clotting and platelet aggregation assays, rAamIGFBP-rP1 did not have any effect. Unlike human IGFBP-rP1, which is controlled by trypsinization, rAamIGFBP-rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick-borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24-48 h of the tick starting to feed makes AamIGFBP-rP1 an attractive target for antitick vaccine development.

Fibroblast growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor receptor tyrosine kinase family. It has been identified as a promising therapeutic target in multiple types of cancer. We have investigated FGFR3 protein expression and FGFR3 gene copy-numbers in a single

Factor IX is an essential blood haemostatic protein, which is apparent from the observation that the absence of functional FIX is associated with the severe bleeding disorder haemophilia B. To achieve its full enzymatic activity, the serine protease precursor factor IX must first be activated into

Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

Insufficient placental growth is a major factor contributing to intrauterine growth retardation in mammals. There is growing evidence that putrescine produced from arginine (Arg) and proline via ornithine decarboxylase is a key regulator of angiogenesis, embryogenesis, as well as placental and fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that putrescine stimulates protein synthesis by activating the mechanistic target of rapamycin (mTOR) signaling pathway in porcine trophectoderm cell line 2 cells. The cells were cultured for 2 to 4 days in customized Arg-free Dulbecco modified Eagle Ham medium containing 0, 10, 25, or 50 μM putrescine or 100 μM Arg. Cell proliferation, protein synthesis, and degradation, as well as the abundance of total and phosphorylated mTOR, ribosomal protein S6 kinase 1, and eukaryotic initiation factor4E-bindingprotein-1 (4EBP1), were determined. Our results indicate that putrescine promotes cell proliferation and protein synthesis in a dose- and time-dependent manner, which was inhibited by difluoro-methylornithine (an inhibitor of ornithine decarboxylase). Moreover, supplementation of culture medium with putrescine increased the abundance of phosphorylated mTOR and its downstream targets, 4EBP1 and p70 S6K1 proteins. Collectively, these findings reveal a novel and important role for putrescine in regulating the mTOR signaling pathway in porcine placental cells. We suggest that dietary supplementation with or intravenous administration of putrescine may provide a new and effective strategy to improve survival and growth of embryos/fetuses in mammals.

Trefoil factor family (TFF), composed of TFF1, TFF2, and TFF3, is a cluster of secreted peptides characterized by trefoil domain (s) and C-terminal dimerization domain. TFF1, a gastric tumor suppressor, is a single trefoil peptide originally detected in breast cancer cell lines but expressed mainly in the stomach; TFF2, a candidate of gastric cancer suppressor with two trefoil domains, is abundant in the stomach and duodenal Brunner's glands; and TFF3 is another single trefoil peptide expressed throughout the intestine which can promote the development of gastric carcinoma. According to multiple studies, TFFs play a regulatory function in the mammals' digestive system, namely in mucosal protection and epithelial cell reconstruction, tumor suppression or promotion, signal transduction and the regulation of proliferation and apoptosis. Action mechanisms of TFFs remain unresolved, but the recent demonstration of a GKN (gastrokine) 2-TFF1 heterodimer implicates structural and functional interplay with gastrokines. This review aims to encapsulate the structural and biological characteristics of TFF.

As more attempts at modulating transcription factor activity are undertaken, valuable knowledge will be accumulated on the modality of action required to modulate transcription and how these findings can be applied to developing transcription factor drugs. Key discoveries will spawn into new therapeutic approaches not only as anticancer targets but also for other indications, such as those with an inflammatory component including neurodegenerative disorders, diabetes, and chronic liver and kidney diseases.

Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.

Background: TGIF2LX (transforming growth factor beta-induced factor 2 like, X-linked) is a homeodomain (HD) protein that has been implicated in the negative regulation of cell signaling pathways. The aim of this study was to investigate the possible functions of TGIF2LX in colon adenocarcinoma cells. Methods: The human SW48 cell line was transfected with cDNA for the wild-type TGIF2LX gene and gene/protein over-expression was confirmed by microscopic analysis, real time RT-PCR and Western blotting techniques. In vitro cell proliferation was evaluated by MTT and BrdU assays. After developing a colon tumor model in nude mice, immunohistochemical (IHC) staining of tumor tissue was carried out for Ki-67 (proliferation) and CD34 (angiogenesis) markers. To predict potential protein partners of TGIF2LX, in-silico analysis was also conducted. Results: Obtained results showed over-expression of TGIF2LX as a potential transcription factor could inhibit either proliferation or angiogenesis (P<0.05) in colon tumors. In-silico results predicted interaction of TGIF2LX with other proteins considered important for cellular development. Conclusions: Our findings provided evidence of molecular mechanisms by which TGIF2LX could act as a tumor suppressor in colon adenocarcinoma cells. Thus, this gene may potentially be a promising option for colon cancer gene-based therapeutic strategies. Creative Commons Attribution License

When mammalian cells are grown in medium containing [3H]spermidine, a single major tritiated protein identical to eukaryotic initiation factor 4D becomes labeled. This protein contains 1 residue/molecule of tritiated hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), a rare amino acid which has been found in no other protein. In order to investigate the conservation of this protein, we examined two nonmammalian eukaryotes, the yeast Saccharomyces cerevisiae and the insect Drosophila melanogaster, and the eubacterial prokaryote Escherichia coli for the presence of the hypusine-containing protein. When the eukaryotic cells were grown in the presence of [3H]spermidine, electrophoretic analysis revealed a single labeled protein. In each case, the apparent molecular weight was near 18,000 and the relative pI was approximately 5.2, similar to the hypusine-containing protein of mammals. Amino acid analysis confirmed the presence of tritiated hypusine in each case, and silver staining of two-dimensional polyacrylamide gels demonstrated that, in yeast and fruit flies as in mammals, the protein is relatively abundant. In the eubacterium E. coli, one tritiated protein was predominant, but its molecular weight was 24,000 and we found no evidence that it contained tritiated hypusine. We found no evidence for the existence of the hypusine-containing protein in the archaebacterium Methanococcus voltae. These data suggest that the hypusine-containing protein is conserved among eukaryotes.

Full Text Available BACKGROUND: Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF, and glial cell line-derived neurotrophic factor (GDNF in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05. In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05. Delivery modes were negatively associated with the concentration of GDNF in human milk. CONCLUSIONS: S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other's nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection.

Protein secondary structural analysis is important for understanding the relationship between protein structure and function, or more importantly how changes in structure relate to loss of function. The structurally sensitive protein vibrational modes (amide I, II, III and S) in deep-ultraviolet resonance Raman (DUVRR) spectra resulting from the backbone C-O and N-H vibrations make DUVRR a potentially powerful tool for studying secondary structure changes. Experimental studies reveal that the position and intensity of the four amide modes in DUVRR spectra of proteins are largely correlated with the varying fractions of α-helix, β-sheet and disordered structural content of proteins. Employing multivariate calibration methods and DUVRR spectra of globular proteins with varying structural compositions, the secondary structure of a protein with unknown structure can be predicted. A disadvantage of multivariate calibration methods is the requirement of known concentration or spectral profiles. Second-order curve resolution methods, such as parallel factor analysis (PARAFAC), do not have such a requirement due to the "second-order advantage." An exceptional feature of DUVRR spectroscopy is that DUVRR spectra are linearly dependent on both excitation wavelength and secondary structure composition. Thus, higher order data can be created by combining protein DUVRR spectra of several proteins collected at multiple excitation wavelengths to give multi-excitation ultraviolet resonance Raman data (ME-UVRR). PARAFAC has been used to analyze ME-UVRR data of nine proteins to resolve the pure spectral, excitation and compositional profiles. A three factor model with non-negativity constraints produced three unique factors that were correlated with the relative abundance of helical, β-sheet and poly-proline II dihedral angles. This is the first empirical evidence that the typically resolved "disordered" spectrum represents the better defined poly-proline II type structure.

We previously reported that zerumbone, a sesquiterpene found in Zingiber zerumbet SMITH, showed notable cancer preventive effects in various organs of experimental rodents. This agent up-regulated nuclear factor-E2-related factor (Nrf2)-dependent expressions of anti-oxidative and xenobiotics-metabolizing enzymes, leading to an increased self-defense capacity. On the other hand, zerumbone markedly suppressed the expression of cyclooxygenase-2, an inducible pro-inflammatory enzyme, by disrupting mRNA stabilizing processes. Binding experiments using a biotin derivative of zerumbone demonstrated that Keap1, an Nrf2 repressive protein, is one of its major binding proteins that promotes their dissociation for inducing Nrf2 transactivation. We then generated a specific antibody against zerumbone-modified proteins and found that zerumbone modified numerous cellular proteins in a non-specific manner, with global distribution of the modified proteins seen not only in cytoplasm but also the nucleus. Based on those observations, zerumbone was speculated to cause proteo-stress, a notion supported by previous findings that it increased the C-terminus of Hsc70 interacting protein-dependent protein ubiquitination and also promoted aggresome formation. Interestingly, zerumbone counteracted proteo-stress and heat stress via up-regulation of the protein quality control systems (PQCs), e.g., heat shock proteins (HSPs), ubiquitin-proteasome, and autophagy. Meanwhile, several phytochemicals, including ursolic acid and curcumin, were identified as marked HSP70 inducers, whereas most nutrients tested were scarcely active. Recent studies have revealed that PQCs play important roles in the prevention of many lifestyle related diseases, such as cancer, thus non-specific binding of phytochemicals to cellular proteins may be a novel and unique mechanism underlying their physiological activities.

A single cellular protein of Mr approximately 18,000 and pI near 5.1, recently identified as eukaryotic translation initiation factor eIF-4D, contains the unusual amino acid hypusine [N epsilon-(4-amino--2-hydroxybutyl)lysine] formed post-translationally from lysine with a structural contribution from the polyamine spermidine. When the 3H-labeled hypusine-containing protein isolated from Chinese hamster ovary (CHO) cells that were grown with radioactive polyamine is digested with trypsin and the digest is subjected to two-dimensional separation, a single radioactive peptide is seen. A labeled peptide that occupies this same position is found in a digest of the [3H]hypusine protein from human lymphocytes and the single hypusine-containing tryptic peptide from purified rabbit reticulocyte eIF-4D also moves to this identical position. Stepwise Edman degradation of the tryptic digest of CHO cell hypusine-protein releases the radioactivity as a single peak in accordance with our earlier evidence for a single hypusine residue per molecule of eIF-4D. The similar patterns of radioactive peptides obtained from tryptic digests of radioiodinated eIF-4D from CHO cells, human lymphocytes, and rabbit reticulocytes suggest a highly conserved primary structure for this protein.

Full Text Available Tumor necrosis factor α-induced protein 8 (TNFAIP8-like protein 1 (TIPE1 was a member of TNFAIP8 family. Previous studies have shown that TIPE1 could induce apoptosis in hepatocellular carcinoma. In this study, we attempted to predict its potential structure. Bioinformatic analysis of TIPE1 was performed to predict its potential structure using the bioinfomatic web services or softwares. The results showed that the amino acid sequences of TIPE1 were well conserved in mammals. No signal peptide and no transmembrane domain existed in human TIPE1. The aliphatic index of TIPE1 was 100.75 and the theoretical pI was 9.57. TIPE1 was a kind of stable protein and its grand average of hydropathicity was -0.108. Various post-translational modifications were also speculated to exist in TIPE1. In addition, the results of Swiss-Model Server and Swiss-Pdb Viewer program revealed that the predicted three-dimensional structure of TIPE1 protein was stable and it may accord with the rule of stereochemistry. TIPE1 was predicted to interact with FBXW5, caspase8 and so on. In conclusion, TIPE1 may be a stable protein with no signal peptide and no transmembrane domain. The bioinformatic analysis of TIPE1 will provide the basis for the further study on the function of TIPE1.

The complement inhibitor factor H (fH) interacts via its seventh short consensus repeat (SCR) domain with multiple ligands including heparin, streptococcal M protein and C-reactive protein (CRP). The aim of this study was to localize the residues in SCR 7 required for these interactions. We initially built a homology model of fH SCR 6-7 using the averaged NMR structures of fH SCR 15-16 and vaccinia control protein SCR 3-4 as templates. Electrostatic potentials of the model's surface demonstrated a co-localization of three clusters of positively charged residues on SCR 7, labeled site A (R369 and K370), site B (R386 and K387) and site C (K392). These residues, localized to the linker region preceding SCR 7 and to the end of a "hypervariable loop" in SCR 7, were systematically replaced with uncharged alanine residues in an fH construct containing SCR 1-7. The resulting proteins were expressed in the methylotrophic yeast, Pichia pastoris. By ELISA analysis we demonstrated: first, that substituting site A inhibited heparin and CRP binding; secondly, that substituting site B inhibited binding to heparin, CRP and M protein; and thirdly, that substituting site C clearly inhibited only heparin binding.

Sex-related infections are a global problem. Such infections may lead to acute or chronic diseases. Chlamydia trachomatis is a dangerous and widespread pathogenicity factor that is not sensitive to conventional drugs and has no obvious symptoms. Protein CPAF is leading factor of pathogenesis. This protein inhibits the signaling pathways of host cell and supports long survival of the pathogen in the host cell. The goal of this work was to review general properties of the proteasome Chlamydia protein CPAF, its functions, and role in pathology. The role of protein CPAF in the anti-chlamydia immune reaction is discussed. The prospects of the development of promising anti-chlamydia vaccine, as well as new effective anti-chlamydia drugs are also discussed.

The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

Full Text Available Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders.

We show here that in several different cell lines protein kinase CK2 and Fas-associated factor 1 (FAF1) exist together in a complex which is stable to high monovalent salt concentration. The CK2/FAF1 complex formation is significantly increased after induction of apoptosis with various DNA damaging...... the view that protein kinase CK2 plays an important role in certain steps of apoptosis....

A label-free aptasensor for platelet-derived growth factor (PDGF) protein is reported. The aptasensor uses mixed self-assembled monolayers (SAMs) composed of a thiol-modified PDGF binding aptamer and 6-mercaptohexanol (MCH) on a gold electrode. The SAMs were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) before and after binding of the protein using [Fe(CN){sub 6}]{sup 3-/4-}, a redox marker ion as an indicator for the formation of a protein-aptamer complex. The CVs at the PDGF modified electrode showed significant differences, such as changes in the peak currents and peak-to-peak separation, before and after binding of the target protein. The EIS spectra, in the form of Nyquist plots, were analyzed with a Randles circuit while the electron transfer resistance R{sub ct} was used to monitor the binding of the target protein. The results showed that, without any modification to the aptamer, the target protein can be recognized effectively at the PDGF binding aptamer SAMs at the electrode surface. Control experiments using non-binding oligonucleotides assembled at the electrode surfaces also confirmed the results and showed that there was no formation of an aptamer-protein complex. The DPV signal at the aptamer functionalized electrode showed a linearly decreased marker ion peak current in a protein concentrations range of 1-40 nM. Thus, label-free detection of PDGF protein at an aptamer modified electrode has been demonstrated.

Gene transcription is regulated through the interactions of RNA polymerase (RNAP) with transcription factors, such as the bacterial sigma proteins. We have devised a new strategy that relies on targeted protein footprinting to make an extensive survey of proximity to the protein surface. This involves attaching cutting reagents randomly to lysine residues on the surface of a protein such as sigma. The lysine-labeled sigma protein is then used to cleave the polypeptide backbones of the RNAP proteins at exposed residues adjacent to the sigma binding site. We used targeted protein footprinting to compare the areas near which sigma(70), sigma(54), sigma(38), sigma(E), NusA, GreA, and omega bind to the protein subunits of Escherichia coli RNAP. The sigma proteins and NusA cut sites in similar regions of the two large RNAP subunits, beta and beta', outlining a common surface. GreA cuts a larger set of sites, whereas omega shows no overlap with the others, cutting only the beta' subunit at a unique location.

Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin

Full Text Available A growing global population, combined with factors such as changing socio-demographics, will place increased pressure on the world’s resources to provide not only more but also different types of food. Increased demand for animal-based protein in particular is expected to have a negative environmental impact, generating greenhouse gas emissions, requiring more water and more land. Addressing this “perfect storm” will necessitate more sustainable production of existing sources of protein as well as alternative sources for direct human consumption. This paper outlines some potential demand scenarios and provides an overview of selected existing and novel protein sources in terms of their potential to sustainably deliver protein for the future, considering drivers and challenges relating to nutritional, environmental, and technological and market/consumer domains. It concludes that different factors influence the potential of existing and novel sources. Existing protein sources are primarily hindered by their negative environmental impacts with some concerns around health. However, they offer social and economic benefits, and have a high level of consumer acceptance. Furthermore, recent research emphasizes the role of livestock as part of the solution to greenhouse gas emissions, and indicates that animal-based protein has an important role as part of a sustainable diet and as a contributor to food security. Novel proteins require the development of new value chains, and attention to issues such as production costs, food safety, scalability and consumer acceptance. Furthermore, positive environmental impacts cannot be assumed with novel protein sources and care must be taken to ensure that comparisons between novel and existing protein sources are valid. Greater alignment of political forces, and the involvement of wider stakeholders in a governance role, as well as development/commercialization role, is required to address both sources of

Protein-based nanoparticles are attractive carriers for drug delivery because they are biodegradable and can be genetically designed. Moreover, modification of protein-based nanoparticles with cell-specific ligands allows for active targeting abilities. Previously, we developed protein nanoparticles comprising genetically engineered elastin-like polypeptides (ELPs) with fused polyaspartic acid tails (ELP-D). Epidermal growth factor (EGF) was displayed on the surface of the ELP-D nanoparticles via genetic design to allow for active cell-targeting abilities. Herein, we focused on the coiled-coil structural motif as a means for noncovalent tethering of growth factor to ELP-D. Specifically, two peptides known to form a heterodimer via a coiled-coil structural motif were fused to ELP-D and single-chain vascular endothelial growth factor (scVEGF121), to facilitate noncovalent tethering upon formation of the heterodimer coiled-coil structure. Drug-loaded growth factor-tethered ELP-Ds were found to be effective against cancer cells by provoking cell apoptosis. These results demonstrate that tethering growth factor to protein nanoparticles through coiled-coil formation yields a promising biomaterial candidate for targeted drug delivery.

(-)-Epigallocatechin gallate (EGCG), a major polyphenolic constituent of green tea, can inhibit activity of specific receptor tyrosine kinases (RTKs) and related downstream signal transduction pathways, resulting in the control of unwanted cell proliferation. The epidermal growth factor receptor (EGFR) signaling pathway is one of the most important pathways that regulates growth, survival, proliferation and differentiation in mammalian cells. This review addresses the effects of EGCG on some proteinfactors involved in the EGFR signaling pathway in a direct or indirect manner. Based on our understanding of the interaction between EGCG and these factors, and based on their structures, EGCG could be used as a lead compound for designing and synthesizing novel drugs with significant biological activity.

Distributions of scaled B factors from 704 protein–DNA complexes reflect primarily the neighbourhood of amino-acid and nucleotide residues: their flexibility grows from the protein core to protein–protein and protein–DNA interfaces, to solvent-exposed residues. Some of the findings clearly observed at higher resolution structures can no longer be observed for structures at low resolution indicating problems in refinement protocols. The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein–DNA crystal structures were analyzed [Schneider et al. (2014 ▶), Nucleic Acids Res.42, 3381–3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein–protein or protein–DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Background Specificity protein (Sp) transcription factors play pivotal roles in maintaining the phenotypes of many cancers. We hypothesized that the antineoplastic effects of sulindac and its metabolites were due, in part, to targeting downregulation of Sp transcription factors. Methods The functional effects of sulindac, sulindac sulfone and sulindac sulfide on colon cancer cell proliferation were determined by cell counting. Effects of these compounds on expression of Sp1, Sp3, Sp4 and pro-...

Insulin-like growth factor binding proteins (IGFBPs) are major regulators of insulin-like growth factor bioavailability and activity in metabolic signaling. Seven IGFBP family isoforms have been identified. Recent studies have shown that IGFBPs play a pivotal role in metabolic signaling and disease, including the pathogenesis of obesity, diabetes, and cancer. Although many studies have documented the various roles played by IGFBPs, transcriptional regulation of IGFBPs is not well understood. ...

An emerging concept in transcriptional regulation is that a class of truncated transcription factors (TFs), called microProteins (miPs), engages in protein-protein interactions with TF complexes and provides feedback controls. A handful of miP examples have been described in the literature but the e

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-bindin

Similar to findings in the nephrotic syndrome in humans, rats with the doxorubicin-induced nephrotic syndrome (which resembles minimal change disease) have reduced serum levels of insulin-like growth factor-I (IGF-I). This is mainly caused by glomerular ultrafiltration of IGF-I-containing binding protein complexes, primarily of a molecular weight of approximately 50 kilodaltons, and urinary losses of the peptide. Despite urinary excretion of IGF-binding protein (IGFBP)-2, serum levels are increased more than twofold in the nephrotic syndrome compared with controls, because of increased synthesis of this binding protein by the liver. In contrast, the liver synthesis of IGFBP-3, the predominant binding protein in normal serum, is unchanged in the nephrotic syndrome. However, binding and serum levels of IGFBP-3 are reduced in nephrotic rat serum, apparently due to proteolytic degradation of IGFBP-3. The glomerular ultrafiltration of IGF-I, which leads to biologically significant IGF-I concentrations of about 1.35 nM in proximal tubule fluid, may have metabolic consequences, such as increased tubular phosphate absorption. Hypothetically, tubule fluid IGF-I may also contribute to progressive tubulointerstitial fibrosis which is sometimes present in protractive nephrotic glomerulopathies. The profound changes in the IGF-I/IGFBP system in the nephrotic syndrome may also contribute to systemic metabolic abnormalities and growth failure.

Full Text Available Lack of tyrosine sulfation of ocular proteins results in disorganized photoreceptor structure and drastically reduced visual function, demonstrating the importance of this post-translational modification to vision. To understand the role that tyrosine sulfation plays in the function of ocular proteins, we identified some tyrosine-sulfated proteins in the retinal pigment epithelium using two independent methods, immuno-affinity column purification with an anti-sulfotyrosine specific antibody and computer-based sequence analysis of retinal pigment epithelium secretome by means of the prediction program Sulfinator. Radioactive labeling followed by thin layer electrophoresis revealed that three proteins, vitronectin, opticin, and complement factor H (CFH, were post-translationally modified by tyrosine sulfation. The identification of vitronectin and CFH as tyrosine-sulfated proteins is significant, since both are deposited in drusen in the eyes of patients with age-related macular degeneration (AMD. Furthermore, mutations in CFH have been determined to be a major risk factor in the development of AMD. Future studies that seek to understand the role of CFH in the development of AMD should take into account the role that tyrosine sulfation plays in the interaction of this protein with its partners, and examine whether modulating sulfation provides a potential therapeutic target.

textabstractInsulin-like growth factors (IGF-I and IGF-II) are single chain polypeptides mediating many of the growth promoting effects of growth hormone, as stated in the "somatomedin hypothesis" (Daughaday et al., 1972). IGF-I is a basic protein and consists of 70 AA with a predicted molecular

In the CNS insulin-like growth factor-1 (IGF-1) enhances survival of neurons, promotes myelin synthesis and acts as a mitogen for microglia. The effects of IGF-1 are regulated by a family of 6 IGF binding proteins (IGFBPs). We investigated mRNA expression patterns of IGFBPs in primary rat microglia

Schistosoma mansoni eggs have to cross the endothelium and intestinal wall to leave the host and continue the life cycle. Mechanisms involved in this essential step are largely unknown. Here we describe direct binding to the S. mansoni eggshell of von Willebrand factor and other plasma proteins invo

Schistosoma mansoni eggs have to cross the endothelium and intestinal wall to leave the host and continue the life cycle. Mechanisms involved in this essential step are largely unknown. Here we describe direct binding to the S. mansoni eggshell of von Willebrand factor and other plasma proteins

textabstractInsulin-like growth factors (IGF-I and IGF-II) are single chain polypeptides mediating many of the growth promoting effects of growth hormone, as stated in the "somatomedin hypothesis" (Daughaday et al., 1972). IGF-I is a basic protein and consists of 70 AA with a predicted molecular wei

Epithelial cell growth regulation has been reported to be altered in inflammatory bowel disease (IBD) patients. The cell growth regulatory factor, insulin-like growth factor binding protein 3 (IGFBP-3), may be partly responsible for this phenomenon. So far, IGFBP-3 levels have been assessed...... as values of total protein, which is a sum of bioactive intact 43- to 45-kDa protein and its inactive proteolytic cleavage fragments. We aimed to assess the levels of intact IGFBP-3 and its cleaving protease MMP-9 in IBD. Patients with IBD and controls were included. Total plasma IGFBP-3 concentration...... and MMP-9 levels were determined in ELISA. The concentration of intact IGFBP-3 was significantly decreased in patients with moderate to severe IBD activity compared to those in remission or controls. Of note, a dramatic depletion of intact IGFBP-3 was found in 7.4% of patients with IBD. Zymography...

Full Text Available Abstract Background The role of the epidermal growth factor receptor (EGFR and other receptor tyrosine kinases (RTKs in provoking biological actions of G protein-coupled receptors (GPCRs has been one of the most disputed subjects in the field of GPCR signal transduction. The purpose of the current study is to identify EGFR-mediated mechanisms involved in activation of G protein cascades and the downstream transcription factors by lysophosphatidic acid (LPA. Results In ovarian cancer cells highly responsive to LPA, activation of AP-1 by LPA was suppressed by inhibition of EGFR, an effect that could be reversed by co-stimulation of another receptor tyrosine kinase c-Met with hepatocyte growth factor, indicating that LPA-mediated activation of AP-1 requires activity of a RTK, not necessarily EGFR. Induction of AP-1 components by LPA lied downstream of Gi, G12/13, and Gq. Activation of the effectors of Gi, but not Gq or G12/13 was sensitive to inhibition of EGFR. In contrast, LPA stimulated another prominent transcription factor NF-κB via the Gq-PKC pathway in an EGFR-independent manner. Consistent with the importance of Gi-elicited signals in a plethora of biological processes, LPA-induced cytokine production, cell proliferation, migration and invasion require intact EGFR. Conclusions An RTK activity is required for activation of the AP-1 transcription factor and other Gi-dependent cellular responses to LPA. In contrast, activation of G12/13, Gq and Gq-elicited NF-κB by LPA is independent of such an input. These results provide a novel insight into the role of RTK in GPCR signal transduction and biological functions.

The segrosome is the nucleoprotein complex that mediates accurate segregation of bacterial plasmids. The segrosome of plasmid TP228 comprises ParF and ParG proteins that assemble on the parH centromere. ParF, which exemplifies one clade of the ubiquitous ParA superfamily of segregation proteins, polymerizes extensively in response to ATP binding. Polymerization is modulated by the ParG centromere binding factor (CBF). The segrosomes of plasmids pTAR, pVT745 and pB171 include ParA homologues of the ParF subgroup, as well as diverse homodimeric CBFs with no primary sequence similarity to ParG, or each other. Centromere binding by these analogues is largely specific. Here, we establish that the ParF homologues of pTAR and pB171 filament modestly with ATP, and that nucleotide hydrolysis is not required for this polymerization, which is more prodigious when the cognate CBF is also present. By contrast, the ParF homologue of plasmid pVT745 did not respond appreciably to ATP alone, but polymerized extensively in the presence of both its cognate CBF and ATP. The co-factors also stimulated nucleotide-independent polymerization of cognate ParF proteins. Moreover, apart from the CBF of pTAR, the disparate ParG analogues promoted polymerization of non-cognate ParF proteins suggesting that filamentation of the ParF proteins is enhanced by a common mechanism. Like ParG, the co-factors may be modular, possessing a centromere-specific interaction domain linked to a flexible region containing determinants that promiscuously stimulate ParF polymerization. The CBFs appear to function as bacterial analogues of formins, microtubule-associated proteins or related ancillary factors that regulate eucaryotic cytoskeletal dynamics.

The relationship between mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis during instances of amino acid surplus in humans is based solely on correlational data. Therefore, the goal of this study was to use a mechanistic approach specifically designed to determine whether increased mTORC1 activation is requisite for the stimulation of muscle protein synthesis following L-essential amino acid (EAA) ingestion in humans. Examination of muscle protein synthesis and signaling were performed on vastus lateralis muscle biopsies obtained from 8 young (25 ± 2 y) individuals who were studied prior to and following ingestion of 10 g of EAA during 2 separate trials in a randomized, counterbalanced design. The trials were identical except during 1 trial, participants were administered a single oral dose of a potent mTORC1 inhibitor (rapamycin) prior to EAA ingestion. In response to EAA ingestion, an ~60% increase in muscle protein synthesis was observed during the control trial, concomitant with increased phosphorylation of mTOR (Ser(2448)), ribosomal S6 kinase 1 (Thr(389)), and eukaryotic initiation factor4Ebindingprotein 1 (Thr(37/46)). In contrast, prior administration of rapamycin completely blocked the increase in muscle protein synthesis and blocked or attenuated activation of mTORC1-signaling proteins. The inhibition of muscle protein synthesis and signaling was not due to differences in either extracellular or intracellular amino acid availability, because these variables were similar between trials. These data support a fundamental role for mTORC1 activation as a key regulator of human muscle protein synthesis in response to increased EAA availability. This information will be useful in the development of evidence-based nutritional therapies targeting mTORC1 to counteract muscle wasting associated with numerous clinical conditions.

HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2 and that overexpression of CBFβ disrupts the interaction between MDM2 and Vif. Knockdown of CBFβ reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ.

The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols.

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic fac

Insects are considered a nutritionally valuable source of alternative proteins, and their efficient protein extraction is a prerequisite for large-scale use. The protein content is usually calculated from total nitrogen using the nitrogen-to-protein conversion factor (Kp) of 6.25. This factor overestimates the protein content, due to the presence of nonprotein nitrogen in insects. In this paper, a specific Kp of 4.76 ± 0.09 was calculated for larvae from Tenebrio molitor, Alphitobius diaperinus, and Hermetia illucens, using amino acid analysis. After protein extraction and purification, a Kp factor of 5.60 ± 0.39 was found for the larvae of three insect species studied. We propose to adopt these Kp values for determining protein content of insects to avoid overestimation of the protein content. PMID:28252948

Full Text Available Molluscum contagiosum virus is a human and animal dermatotropic pathogen, which causes a severe disease in immunocompromised individuals. MCV belongs to the Poxviridae family whose members exert immunomodulatory effects on the host antiviral response. Poxviruses interfere with cell signaling pathways that lead to the activation of nuclear factor кB, a pleiotropic transcription factor which is crucial for regulation of the immune response, the cell cycle and apoptosis. In resting cells, NF-κB is present in the cytoplasm, where it is associated with inhibitor κB. Upon stimulation by activators, such as proinflammatory cytokines and bacterial or viral products, the inhibitory protein undergoes phosphorylation and proteasomal degradation. NF-κB, in turn, translocates to the nucleus, where it regulates the transcription of various genes that are essential for processes mentioned above. Since poxviruses replicate exclusively in the cell cytoplasm, NF-кB became a good target for poxviral immunomodulation. MCV encodes various proteins which interfere with the signaling pathways that lead to the activation of NF-κB. Ligand inhibitor encoded by MCV, MC54, binds interleukin-18 and inhibits interferon-γ production. Other MCV proteins, MC159 and MC160, belong to intracellular inhibitors of NF-κB and are members of viral FLICE-inhibitory proteins (vFLIPs. MC159 protein encoded by MCV was shown to inhibit apoptosis of virus-infected cells. Such interactions serve immune evasion and are responsible for the persistence of MCV.

Full Text Available Abstract Background Insulin-like growth factors (IGFs are important for the proliferation of cancer cells. One of their binding proteins, known as insulin-like growth factor binding protein -4 (IGFBP-4 is well known for its inhibitory action on IGFs in vitro. We assessed the effect of IGFBP-4 in prevention of development of colon cancer in vivo. Methods Nude mice were subcutaneously inoculated with HT-29 colon cancer cells and they were also simultaneously injected either gene construct containing mammalian expression vector pcDNA3 with or without IGFBP-4 gene or phosphate buffered saline. The effect was assessed 4 weeks later by evaluating the tumours for mitosis, necrosis, apoptosis, and expressions of IGFBP-4, Bcl-2 and Bax proteins. Results The results showed that the IGFBP-4 gene therapy did not prevent the tumour establishment but it increased the tumour apoptosis which was associated with an increase in Bcl-2 and Bax expressions. The IGFBP-4 protein was low in tumours which received IGFBP-4 gene construct which may be due to a feed back mechanism of IGFBP-4 upon its own cells. Conclusion IGFBP-4 gene therapy in the form localised gene transfer did not prevent colon cancer initiation and establishment but it resulted in increased apoptosis and Bax protein expression and a decrease in tumour cellular mitosis

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable. PMID:28202722

Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

IgA is the predominant Ig isotype in mucosal secretions and thus plays a pivotal role in host defense. The mechanisms by which IgA expression is regulated may differ among species and involve multiple pathways. Various cytokines and costimulators have been identified which regulate expression of this isotype, including IL-10, IL-2, vasoactive intestinal peptide, and TGF-beta. We have tested a wide array of known factors, but only under very limited conditions do these factors mediate substantial IgA production in vitro from bovine B cells. In response to these findings, we generated a cDNA library in a mammalian expression vector from activated cells derived from bovine gut-associated lymphoid tissues (Peyer's patch and mesenteric lymph node cells) as a source of soluble factor(s) that may regulate IgA production. We have identified a novel factor, IgA-inducing protein, which stimulates relatively high levels of IgA production in vitro following CD40 stimulation in coculture with IL-2. Our data suggest that IgA-inducing protein regulates IgA by acting as a switch or differentiation factor and is expressed in a variety of lymphoid and nonlymphoid tissues.

DOCK (dedicator of cytokinesis) guanine nucleotide exchange factors (GEFs) activate the Rho-family GTPases Rac and Cdc42 to control cell migration, morphogenesis, and phagocytosis. The DOCK A and B subfamilies activate Rac, whereas the DOCK D subfamily activates Cdc42. Nucleotide exchange is catalyzed by a conserved DHR2 domain (DOCK(DHR2)). Although the molecular basis for DOCK(DHR2)-mediated GTPase activation has been elucidated through structures of a DOCK9(DHR2)-Cdc42 complex, the factors determining recognition of specific GTPases are unknown. To understand the molecular basis for DOCK-GTPase specificity, we have determined the crystal structure of DOCK2(DHR2) in complex with Rac1. DOCK2(DHR2) and DOCK9(DHR2) exhibit similar tertiary structures and homodimer interfaces and share a conserved GTPase-activating mechanism. Multiple structural differences between DOCK2(DHR2) and DOCK9(DHR2) account for their selectivity toward Rac1 and Cdc42. Key determinants of selectivity of Cdc42 and Rac for their cognate DOCK(DHR2) are a Phe or Trp residue within β3 (residue 56) and the ability of DOCK proteins to exploit differences in the GEF-induced conformational changes of switch 1 dependent on a divergent residue at position 27. DOCK proteins, therefore, differ from DH-PH GEFs that select their cognate GTPases through recognition of structural differences within the β2/β3 strands.

Studies were conducted to investigate the effects of pH value, extraction time, extraction temper- ature, ratio of material to extraction medium and salt concentration on the solubility of yellow mealworm larvae protein, Tne results showed that pH value greatly influenced the solubility, minimum solubility was found at pH 5.8 and a maximum at pH 10 - 12. Salt ion could increase the solubility at the iso-electric point pH and slightly decreased the solubility at the pH value higher than that of the iso-electric point. Other factors did not significantly affect the solubility of the protein.

Corticotropin-releasing factor (CRF) binding protein (CRF-BP) is a secreted protein that acts to bind and limit the activity of the neuropeptides, CRF and urocortin (Ucn) 1. We previously selected for high maternal defense (protection of offspring) in mice and found CRF-BP to be elevated in the CNS of selected mice. We also previously determined that both CRF and Ucn 1 are potent inhibitors of offspring protection when administered centrally. Thus, elevated CRF-BP could promote defense by lim...

Accurately determining protein content is essential in exploiting biomass as feed and fuel. A survey of biomass samples in China indicated protein contents from 2.65 to 3.98 % for crop residues and from 6.07 to 10.24 % for animal manure. Conversion factors based on amino acid nitrogen (kA) ranged from 5.42 to 6.00 for the former and from 4.78 to 5.36 for the latter, indicating that the traditional factor of 6.25 is not suitable for biomass samples. On the other hand, conversion factors from Kjeldahl nitrogen (kP) ranged from 3.97 to 4.57 and from 2.76 to 4.31 for crop residues and animal manure, respectively. Of note, conversion factors were strongly affected by amino acid composition and levels of nonprotein nitrogen. Thus, kP values of 4.23 for crop residues, 4.11 for livestock manure, and 3.11 for poultry manure are recommended to better estimate protein content from total nitrogen.

Meningococcal factor H binding protein (fHbp) is a human species-specific ligand for the complement regulator, factor H (fH). In recent studies, fHbp vaccines in which arginine at position 41 was replaced by serine (R41S) had impaired fH binding. The mutant vaccines elicited bactericidal responses in human fH transgenic mice superior to those elicited by control fHbp vaccines that bound human fH. Based on sequence similarity, fHbp has been classified into three variant groups. Here we report that although R41 is present in fHbp from variant groups 1 and 2, the R41S substitution eliminated fH binding only in variant group 1 proteins. To identify mutants in variant group 2 with impaired fH binding, we generated fHbp structural models and predicted 63 residues influencing fH binding. From these, we created 11 mutants with one or two amino acid substitutions in a variant group 2 protein and identified six that decreased fH binding. Three of these six mutants retained conformational epitopes recognized by all six anti-fHbp monoclonal antibodies (MAbs) tested and elicited serum complement-mediated bactericidal antibody titers in wild-type mice that were not significantly different from those obtained with the control vaccine. Thus, fHbp amino acid residues that affect human fH binding differ across variant groups. This result suggests that fHbp sequence variation induced by immune selection also affects fH binding motifs via coevolution. The three new fHbp mutants from variant group 2, which do not bind human fH, retained important epitopes for eliciting bactericidal antibodies and may be promising vaccine candidates.

Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3. DOI: http://dx.doi.org/10.7554/eLife.16673.001 PMID:27532772

Full Text Available 14-3-3 proteins are regulatory proteins found in all eukaryotes and are known to selectively interact with phosphorylated proteins to regulate physiological processes. Through an affinity purification screening, many light-related proteins were recovered as 14-3-3 candidate binding partners. Yeast two-hybrid analysis revealed that the 14-3-3 kappa isoform (14-3-3κ could bind to PHYTOCHROME INTERACTING FACTOR3 (PIF3 and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1. Further analysis by in vitro pull-down assay confirmed the interaction between 14-3-3κ and PIF3. Interruption of putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 was not sufficient to inhibit 14-3-3κ from binding or to disturb nuclear localization of PIF3. It was also indicated that 14-3-3κ could bind to other members of the PIF family, such as PIF1 and PIF6, but not to LONG HYPOCOTYL IN FAR-RED1 (HFR1. 14-3-3 mutants, as well as the PIF3 overexpressor, displayed longer hypocotyls, and a pif3 mutant displayed shorter hypocotyls than the wild-type in red light, suggesting that 14-3-3 proteins are positive regulators of photomorphogenesis and function antagonistically with PIF3. Consequently, our results indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to a light signal.

Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years.

The alpha-subunit of the human eukaryotic initiation factor 2 (heIF2alpha), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2alpha gets phosphorylated by eIF2alpha-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2alpha, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (tau(1)=1.13ns and tau(2)=4.74ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the alpha-helix and beta-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted alpha-helix and rearranged beta-sheet in presence of 4M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 degrees C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions.

Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor gene family members play key roles in diverse aspects of plant development. LBD10 and LBD27 have been shown to be essential for pollen development in Arabidopsis thaliana. From the previous RNA sequencing (RNA-Seq) data set of Arabidopsis pollen, we identified the mRNAs of LBD22, LBD25 and LBD36 in addition to LBD10 and LBD27 in Arabidopsis pollen. Here we conducted expression and cellular analysis using GFP:GUS (green fluorescent protein:β-glucuronidase) reporter gene and subcellular localization assays using LBD:GFP fusion proteins expressed under the control of their own promoters in Arabidopsis. We found that these LBD proteins display spatially and temporally distinct and overlapping expression patterns during pollen development. Bimolecular fluorescence complementation and GST (glutathione S-transferase) pull-down assays demonstrated that protein-protein interactions occur among the LBDs exhibiting overlapping expression during pollen development. We further showed that LBD10, LBD22, LBD25, LBD27 and LBD36 interact with each other to form heterodimers, which are localized to the nucleus in Arabidopsis protoplasts. Taken together, these results suggest that combinatorial interactions among LBD proteins may be important for their function in pollen development in Arabidopsis.

Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications.

PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor4E-bindingprotein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona–NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13 nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications. - Highlights: • Dynamic flow increased the size of the gold nanoparticle protein corona. • Exposure to biological fluids altered protein corona size and composition. • Interstitial fluid modified the nano-cellular interface and deposition efficiency. • Tannic acid coated nanoparticles induced toxicity in an interstitial environment.

The oncogenic transcription factor STAT3 is inappropriately activated in multiple hematopoietic and solid malignancies, in which it drives the expression of genes involved in cell proliferation, differentiation, survival, and angiogenesis. Thus far, strategies to inhibit the function of STAT3 have focused on blocking the function of its activating kinases or sequestering its DNA binding ability. A less well-explored aspect of STAT3 function is its interaction with other proteins, which can modulate the oncogenic activity of STAT3 via its subcellular localization, DNA binding ability, and recruitment of transcriptional machinery. Herein we summarize what is currently known about STAT3-interacting proteins and describe the utility of a proteomics-based approach for successfully identifying and characterizing novel STAT3-interacting proteins that affect STAT3 transcriptional activity and oncogenic function.

Hybrid protein, cancer necrosis factor thymosin-alpha1 (CNF-T), when synthesizing in strain-producer of Escherichia coli SG200-50 with plasmid pThy315, was a part of "inclusion bodies" mostly in the form of a high-molecular complex with other proteins due to the S-S bonds formation. An approach of purification of CNF-T has been proposed, which is based on the destruction of the complex in the presence of sodium dodecylsulfate (DDS-NA) and dithiotreitol (DDT) followed by gel-filtration on Sephadex G-100 and renaturation by ultrafiltration on hollow fibers. The method allows the isolation of electrophoretically homogeneous CNF-T containing no DDS-Na and having high cytotoxic activity against cancer cells of mouse adenocarcinome L-929. The yield of CNF-T achieved 80% relative its content in biomass and 30% relative the total protein.

The Polycomb (Pc) group (Pc-G) of repressors is essential for transcriptional silencing of homeotic genes that determine the axial development of metazoan animals. It is generally believed that the multimeric complexes formed by these proteins nucleate certain chromatin structures to silence promoter activity upon binding to Pc-G response elements (PRE). Little is known, however, about the molecular mechanism involved in sequence-specific binding of these complexes. Here, we show that an immunoa ffinity-purified Pc protein complex contains a DNA binding activity specific to the (GA), motif in a PRE from the bithoraxoid region. We found that this activity can be attributed primarily to the large protein isoform encoded by pipsqueak (psq) instead of to the well-characterized GAGA factor. The functional relevance ofpsq to the silencing mechanismis strongly supported by its synergistic interactions with a subset of Pc-G that cause misexpression of homeotic genes.

In the field of tissue engineering, the tethering of growth factors to tissue scaffolds in an oriented manner can enhance their activity and increase their half-life. We chose to investigate the capture of the basic Fibroblast Growth Factor (bFGF) and the Epidermal Growth Factor (EGF) on a gelatin layer, as a model for the functionalization of collagen-based biomaterials. Our strategy relies on the use of two high affinity interactions, that is, the one between two distinct coil peptides as well as the one occurring between a collagen-binding domain (CBD) and gelatin. We expressed a chimeric protein to be used as an adaptor that comprises one of the coil peptides and a CBD derived from the human fibronectin. We proved that it has the ability to bind simultaneously to a gelatin substrate and to form a heterodimeric coiled-coil domain with recombinant growth factors being tagged with the complementary coil peptide. The tethering of the growth factors was characterized by ELISA and surface plasmon resonance-based biosensing. The bioactivity of the immobilized bFGF and EGF was evaluated by a human umbilical vein endothelial cell proliferation assay and a vascular smooth muscle cell survival assay. We found that the tethering of EGF preserved its mitogenic and anti-apoptotic activity. In the case of bFGF, when captured via our adaptor protein, changes in its natural mode of interaction with gelatin were observed.

Suicide is a major public health concern. Although there have been several studies of suicidal behavior that focused on the roles of psychosocial and sociocultural factors, these factors are of too little predictive value to be clinically useful. Therefore, research on the biological perspective of suicide has gained a stronghold and appears to provide a promising approach to identify biological risk factors associated with suicidal behavior. Recent studies demonstrate that an alteration in synaptic and structural plasticity is key to affective illnesses and suicide. Signal transduction molecules play an important role in such plastic events. Protein kinase A (PKA) is a crucial enzyme in the adenylyl cyclase signal transduction pathway and is involved in regulating gene transcription, cell survival, and plasticity. In this review, we critically and comprehensively discuss the role of PKA in suicidal behavior. Because stress is an important component of suicide, we also discuss whether stress affects PKA and how this may be associated with suicidal behavior. In addition, we also discuss the functional significance of the findings regarding PKA by describing the role of important PKA substrates (i.e., Rap1, cyclic adenosine monophosphate response element binding protein, and target gene brain-derived neurotrophic factor). These studies suggest the interesting possibility that PKA and related signaling molecules may serve as important neurobiological factors in suicide and may be relevant in target-specific therapeutic interventions for these disorders.

A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.

Full Text Available The transcription factor AP-2ε (activating enhancer-binding protein epsilon is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4 strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1, the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2′-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

Full Text Available Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1 interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5% followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.

Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4Ebindingprotein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type-dependent manner, with 4E-BP1 being a key player.

The biological activity and functional specificity of proteins depend on their native three-dimensional structures determined by inter- and intra-molecular interactions. In this paper, we investigate the geometrical factor of protein conformation as a consequence of energy minimization in protein folding. Folding simulations of 10 polypeptides with chain length ranging from 183 to 548 residues manifest that the dimensionless ratio (V/(A)) of the van der Waals volume V to the surface area A and average atomic radius of the folded structures, calculated with atomic radii setting used in SMMP [Eisenmenger F., et. al., Comput. Phys. Commun., 138 (2001) 192], approach 0.49 quickly during the course of energy minimization. A large scale analysis of protein structures show that the ratio for real and well-designed proteins is universal and equal to 0.491\\pm0.005. The fractional composition of hydrophobic and hydrophilic residues does not affect the ratio substantially. The ratio also holds for intrinsically disorde...

OBJECTIVE To compare the expression of the thyroid transcription factor-1 (TTF-1) in human normal adult type Ⅱ alveolar epithelial cells,embryonic pneumocytes and cancer cells of lung carcinoma and metastatic lymph nodes using a tissue microarray (TMA) along with paired conventional full sections.and to jnvestigate the reliability of tissue microarrays in detecting protein expression in lung carcinoma.METHODS A lung carcinoma TMA including 765 cores was constructed.TTF-1 protein expression in both TMA and paired conventional full sections were detected by yhe immunohistochemical SP method using a monoclonal antibody to TTF-1.A PU (Positive Unit) of TTF-1 protein was assessed quantitatively by the Leica Q500MC image analysis system with results from the paired conventional full sections as controls.RESULTS There was no signifcance between TMA and paired conven tional full sections in TTF-1 expression in difierent nuclei of the lung tissue.CONCLUSION TTF-1 protein expression in lung carcinoma detected by TMA was highly concordanl with that of paired full sections.TMA is a reliable method in detecting protein expression.

We study the dynamic structure factor S(k,t) of proteins at large wave numbers k, kRg≫1, where Rg is the gyration radius. At this regime measurements are sensitive to internal dynamics, and we focus on vibrational dynamics of folded proteins. Exploiting the analogy between proteins and fractals, we perform a general analytic calculation of the displacement two-point correlation functions, . We confront the derived expressions with numerical evaluations that are based on protein data bank (PDB) structures and the Gaussian network model (GNM) for a few proteins and for the Sierpinski gasket as a controlled check. We use these calculations to evaluate S(k,t) with arrested rotational and translational degrees of freedom, and show that the decay of S(k,t) is dominated by the spatially averaged mean-square displacement of an amino acid. The latter has been previously shown to evolve subdiffusively in time, ˜tν, where ν is the anomalous diffusion exponent that depends on the spectral dimension ds and fractal dimension df. As a result, for wave numbers obeying k2≳1, S(k,t) effectively decays as a stretched exponential S(k,t)≃S(k)e-(Γkt)β with β≃ν, where the relaxation rate is Γk˜(kBT/mωo2)1/βk2/β, T is the temperature, and mωo2 the GNM effective spring constant describing the interaction between neighboring amino acids. The static structure factor is dominated by the fractal character of the native fold, S(k)˜k-df, with negligible to marginal influence of vibrations. The analytical expressions are first confronted with numerically based calculations on the Sierpinski gasket, and very good agreement is found between simulations and theory. We then perform PDB-GNM-based numerical calculations for a few proteins, and an effective stretched exponential decay of the dynamic structure factor is found, albeit their relatively small size. However, when rotational and translational diffusion are added, we find that their contribution is never negligible due to

Full Text Available Hundreds of double homeobox (DUX genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD. In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain and DUX1 (which is limited to the double homeodomain. Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs. Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs

Full Text Available The aryl hydrocarbon receptor (AHR is a transcription factor and environmental sensor that regulates expression of genes involved in drug-metabolism and cell cycle regulation. Chromatin immunoprecipitation analyses, Ahr ablation in mice and studies with orthologous genes in invertebrates suggest that AHR may also play a significant role in embryonic development. To address this hypothesis, we studied the regulation of Ahr expression in mouse embryonic stem cells and their differentiated progeny. In ES cells, interactions between OCT3/4, NANOG, SOX2 and Polycomb Group proteins at the Ahr promoter repress AHR expression, which can also be repressed by ectopic expression of reprogramming factors in hepatoma cells. In ES cells, unproductive RNA polymerase II binds at the Ahr transcription start site and drives the synthesis of short abortive transcripts. Activation of Ahr expression during differentiation follows from reversal of repressive marks in Ahr promoter chromatin, release of pluripotency factors and PcG proteins, binding of Sp factors, establishment of histone marks of open chromatin, and engagement of active RNAPII to drive full-length RNA transcript elongation. Our results suggest that reversible Ahr repression in ES cells holds the gene poised for expression and allows for a quick switch to activation during embryonic development.

The aryl hydrocarbon receptor (AHR) is a transcription factor and environmental sensor that regulates expression of genes involved in drug-metabolism and cell cycle regulation. Chromatin immunoprecipitation analyses, Ahr ablation in mice and studies with orthologous genes in invertebrates suggest that AHR may also play a significant role in embryonic development. To address this hypothesis, we studied the regulation of Ahr expression in mouse embryonic stem cells and their differentiated progeny. In ES cells, interactions between OCT3/4, NANOG, SOX2 and Polycomb Group proteins at the Ahr promoter repress AHR expression, which can also be repressed by ectopic expression of reprogramming factors in hepatoma cells. In ES cells, unproductive RNA polymerase II binds at the Ahr transcription start site and drives the synthesis of short abortive transcripts. Activation of Ahr expression during differentiation follows from reversal of repressive marks in Ahr promoter chromatin, release of pluripotency factors and PcG proteins, binding of Sp factors, establishment of histone marks of open chromatin, and engagement of active RNAPII to drive full-length RNA transcript elongation. Our results suggest that reversible Ahr repression in ES cells holds the gene poised for expression and allows for a quick switch to activation during embryonic development.

Full Text Available Creatine kinase (CK; EC 2.7.3.2 is related to several skin diseases such as psoriasis and dermatomyositis. CK is important in skin energy homeostasis because it catalyzes the reversible transfer of a phosphoryl group from MgATP to creatine. In this study, we predicted CK binding proteins via the use of bioinformatic tools such as protein-protein interaction (PPI mappings and suggest the putative hub proteins for CK interactions. We obtained 123 proteins for brain type CK and 85 proteins for muscle type CK in the interaction networks. Among them, several hub proteins such as NFKB1, FHL2, MYOC, and ASB9 were predicted. Determination of the binding factors of CK can further promote our understanding of the roles of CK in physiological conditions.

Full Text Available Abstract Background Homeodomain-interacting protein kinase-2 (HIPK2 plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF levels (a β-catenin target gene and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches. Methods The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting. Results HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β activities. Conclusion These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches.

Previous studies have found that estrogen enhances the effect of insulin-like growth factor-I (IGF-I) levels on breast cancer cell growth. Participants in the Shanghai Breast Cancer Study (SBCS) consumed large amounts of soy that was high in isoflavones, which act as weak estrogens and as anti-estrogens. We assessed whether soy protein intake modified the effect of IGF-I levels on breast cancer risk. The SBCS is a population-based case-control study of breast cancer among women aged 25-64 conducted between 1996 and 1998 in urban Shanghai. In-person interviews were completed with 1,459 incident breast cancer cases ascertained through a population-based cancer registry and 1,556 controls randomly selected from the general population (with respective response rates of 91% and 90%). This analysis is restricted to the 397 cases and 397 matched controls for whom information on IGF-I levels was available. For premenopausal breast cancer, we found nearly significant interactions between soy protein intake and IGF-I levels (P = 0.080) and insulin-like growth factor-binding protein-3 (IGFBP-3) levels (P = 0.057). The direction of the interaction appeared to be negative for IGF-I levels but was positive for IGFBP-3 levels. No interaction was evident between soy protein intake and IGF-I or IGFBP-3 levels among postmenopausal women. Our results suggest that soy protein intake may negatively modulate the effect of IGF-I and may positively modulate the effect of IGFBP-3 levels on premenopausal breast cancer risk. Further studies are needed to confirm our finding and to understand the biological mechanisms of these potential interactions.

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Full Text Available Background: Activated protein C (APC inactivates factor V (FV by cleavage of its heavy chain at Arg306, Arg506, Arg679, and Lys994. Mutational changes, which abolish APC cleavage sites, may predispose thrombosis by altering the inactivation process of FV. FV Leiden (FVL (Arg506Glu has been demonstrated as a strong risk factor for thrombosis. In the current study, we have studied whether mutations in the cleavage sites of FV for APC, not due to FVL, would have a role in presenting APC resistance (APCR and initiation of a cerebral thrombotic event.Methods: A group of 22 patients with a history of cerebral venous thrombosis (CVT, who were not carriers of FVL enrolled in the study. The patients who had conditions associated with acquired APCR were excluded from the study. APCR test was performed on the remaining 16 patients, which showed APCR in 4 plasma samples. DNA sequencing was performed on four exons of FV of APCR patients, encoding Arg306, Arg506, Arg679, and Lys994.Results: Mutations were not found within nucleotides encoding the cleavage sites; neither was found within their close upstream and downstream sequences.Conclusion: Our results show that polymorphisms affecting cleavage sites of FV other than Arg506Glu it would be less likely to be the basis for APCR and its increased thrombosis susceptibility. In addition, it emphasizes on the importance of screening for APCR in the patients diagnosed with CVT.

Exercise is increasingly recognized as an intervention that can reduce CNS dysfunctions such as cognitive decline, depression and stress. Previously we have demonstrated that brain-derived neurotrophic factor (BDNF) is increased in the hippocampus following exercise. In this study we tested the hypothesis that exercise can counteract a reduction in hippocampal BDNF protein caused by acute immobilization stress. Since BDNF expression is suppressed by corticosterone (CORT), circulating CORT levels were also monitored. In animals subjected to 2 h immobilization stress, CORT was elevated immediately following, and at 1 h after the cessation of stress, but remained unchanged from baseline up to 24 h post-stress. The stress protocol resulted in a reduction in BDNF protein at 5 and 10 h post-stress that returned to baseline at 24 h. To determine if exercise could prevent this stress-induced reduction in BDNF protein, animals were given voluntary access to running wheels for 3 weeks prior to the stress. Stressed animals, in the absence of exercise, again demonstrated an initial elevation in CORT (at 0 h) and a subsequent decrease in hippocampal BDNF at the 10 h time point. Exercising animals, both non-stressed and stressed, demonstrated circulating CORT and hippocampal BDNF protein levels that were significantly elevated above control values at both time points examined (0 and 10 h post-stress). Thus, the persistently high CORT levels in exercised animals did not affect the induction of BDNF with exercise, and the effect of immobilization stress on BDNF protein was overcome. To examine the role of CORT in the stress-related regulation of BDNF protein, experiments were carried out in adrenalectomized (ADX) animals. BDNF protein was not downregulated as a result of immobilization stress in ADX animals, while there continued to be an exercise-induced upregulation of BDNF. This study demonstrates that CORT modulates stress-related alterations in BDNF protein. Further, exercise

Neospora caninum, of the Apicomplexa phylum, is a common cause of abortions in cattle and nervous system dysfunction in dogs. Rhoptry proteins of Apicomplexa play an important role in virulence. The objectives of this study were to study functions of NcROP5 in N. caninum by deleting the NcROP5 gene from the wild Nc-1 strain. We selected NcROP5 in ToxoDB and successfully constructed an NcROP5 gene-deleted vector, pTCR-NcROP5-CD KO. Then we screened the NcROP5 knockout strains (ΔNcROP5) at the gene, protein and transcription levels. Plaque assay, host cell invasion assay and intracellular proliferation test showed that the ΔNcROP5 strain had less plaque space, weakened invasion capacity and slower intracellular growth. Animal testing showed significantly lower cerebral load of ΔNcROP5 than the load of the Nc-1 strain, as well as a loss of virulence for the ΔNcROP5 strains. Phenotypic analyses using the label-free LC-MS/MS assay-based proteomic method and KEGG pathway enrichment analysis showed a reduction of NcGRA7 transcription and altered expression of multiple proteins including the apicomplexan family of binding proteins. The present study indicated that ROP5 is a key virulence factor in N. caninum in mice. The proteomic profiling of Nc-1 and ΔNcROP5 provided some data on differential proteins. These data provide a foundation for future research of protein functions in N. caninum. PMID:28326073

Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic models with strong repressors.

Full Text Available Abstract Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusion Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic

Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo.

Influenza A virus genomic segments eight codes for non-structural 1 (NS1) protein that is involved in evasion of innate antiviral response, and nuclear export protein (NEP) that participates in the export of viral ribonucleoprotein (RNP) complexes, transcription and replication. Tumor necrosis factor alpha (TNF-α) is highly expressed during influenza virus infections and is considered an anti-infective cytokine. NS1 and NEP proteins were overexpressed and their role on TNF-α expression was evaluated. Both TNF-α mRNA and protein increased in cells transfected with NEP but not with NS1. We further investigate if NS1 or NEP regulates the activity of TNF-α promoter. In the presence of NEP the activity of TNF-α promoter increased significantly compared with the control (83.5±2.9 vs. 30.9±2.8, respectively; p=0.001). This effect decreased 15-fold when the TNF-α promoter distal region was deleted, suggesting the involvement of mitogen-activated protein kinases (MAPK) and NF-kB response elements. This was corroborated by testing the effect produced on TNF-α promoter by the treatment with Raf/MEK/ERK (U0126), NF-kB (Bay-11-7082) and PI3K (Ly294-002) cell signaling inhibitors. Treatment with U0126 and Bay-117082 reduced the activity of TNF-α promoter mediated by NEP (41.5±3.2, 70% inhibition; and 80.6±7.4, 35% inhibition, respectively) compared to mock-treated control. The results suggest a new role for NEP protein that participates in the transcriptional regulation of human TNF-α expression.

Eosinophil cationic protein (ECP) is a major component of eosinophil granule protein that is used as a clinical bio-marker for asthma and allergic inflammatory diseases. Previously, it has been reported that the signal peptide of human ECP (ECPsp) inhibits the cell growth of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), but not mammalian A431 cells. The inhibitory effect is due to the lack of human signal peptide peptidase (hSPP), a protease located on the endoplasmic reticulum (ER) membrane, in the lower organisms. In this study, we show that the epidermal growth factor receptor (EGFR) is upregulated by the exogenous ECPsp-eGFP as a result of the increased expression of the transforming growth factor-alpha (TGF-alpha) at both transcriptional and translational levels in A431 and HL-60 clone 15 cell lines. Furthermore, the N-terminus of ECPsp fragment generated by the cleavage of hSPP (ECPspM1-G17) gives rise to over threefold increase of TGF-alpha protein expression, whereas another ECPsp fragment (ECPspL18-A27) and the hSPP-resistant ECPsp (ECPspG17L) do not show similar effect. Our results indicate that the ECPspM1-G17 plays a crucial role in the upregulation of TGF-alpha, suggesting that the ECPsp not only directs the secretion of mature ECP, but also involves in the autocrine system.

The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria. PMID:28276514

Primordial follicle formation and the subsequent transition of follicles to the primary and secondary stages encompass the early events during folliculogenesis in mammals. These processes establish the ovarian follicle pool and prime follicles for entry into subsequent growth phases during the reproductive cycle. Perturbations during follicle formation can affect the size of the primordial follicle pool significantly, and alterations in follicle transition can cause follicles to arrest at immature stages or result in premature depletion of the follicle reserve. Determining the molecular events that regulate primordial follicle formation and early follicle growth may lead to the development of new fertility treatments. Over the last decade, many of the growth factors and signaling proteins that mediate the early stages of folliculogenesis have been identified using mouse genetic models, in vivo injection studies, and ex vivo organ culture approaches. These studies reveal important roles for the transforming growth factor beta (TGF-beta) superfamily of proteins in the ovary. This article reviews these roles for TGF-beta family proteins and focuses in particular on work from our laboratories on the functions of activin in early folliculogenesis.

Plants contain β-amylase-like proteins (BAMs; enzymes usually associated with starch breakdown) present in the nucleus rather than targeted to the chloroplast. They possess BRASSINAZOLE RESISTANT1 (BZR1)-type DNA binding domains--also found in transcription factors mediating brassinosteroid (BR) responses. The two Arabidopsis thaliana BZR1-BAM proteins (BAM7 and BAM8) bind a cis-regulatory element that both contains a G box and resembles a BR-responsive element. In protoplast transactivation assays, these BZR1-BAMs activate gene expression. Structural modeling suggests that the BAM domain's glucan binding cleft is intact, but the recombinant proteins are at least 1000 times less active than chloroplastic β-amylases. Deregulation of BZR1-BAMs (the bam7bam8 double mutant and BAM8-overexpressing plants) causes altered leaf growth and development. Of the genes upregulated in plants overexpressing BAM8 and downregulated in bam7bam8 plants, many carry the cis-regulatory element in their promoters. Many genes that respond to BRs are inversely regulated by BZR1-BAMs. We propose a role for BZR1-BAMs in controlling plant growth and development through crosstalk with BR signaling. Furthermore, we speculate that BZR1-BAMs may transmit metabolic signals by binding a ligand in their BAM domain, although diurnal changes in the concentration of maltose, a candidate ligand produced by chloroplastic β-amylases, do not influence their transcription factor function.

Activation of blood plasma coagulation in vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. The only plausible explanation consistent with current understanding of coagulation-cascade biochemistry is that procoagulant stimulus arising from the activation complex of the intrinsic pathway is dependent on activator surface area. And yet, it is herein shown that activation of the blood zymogen factor XII (Hageman factor, FXII) dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a “mechanochemical” reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results of this study strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway. PMID:23117212

Background:Psoriasis is a common immune-mediated inflammatory dermatosis.Generalized pustular psoriasis (GPP) is the severe and rare type of psoriasis.The association between tumor necrosis factor-alpha induced protein 3 interacting protein 1 (TNIP1) gene and psoriasis was confirmed in people with multiple ethnicities.This study was to investigate the association between TNIP1 gene polymorphisms and pustular psoriasis in Chinese Han population.Methods:Seventy-three patients with GPP,67 patients with palmoplantar pustulosis (PPP),and 476 healthy controls were collected from Chinese Han population.Six single nucleotide polymorphisms (SNPs) of the TNIP1 gene,namely rs3805435,rs3792798,rs3792797,rs869976,rs17728338,and rs999011 were genotyped by using polymerase chain reaction-ligase detection reaction.Statistical analyses were performed using the PLINK 1.07 package.Allele frequencies and genotyping frequencies for six SNPs were compared by using Chi-square test,odd ratio (OR) (including 95％ confidence interval) were calculated.The haplotype analysis was conducted by Haploview software.Results:The frequencies of alleles of five SNPs were significantly different between the GPP group and the control group (P≤ 7.22 × 10-3),especially in the GPP patients without psoriasis vulgaris (PsV).In the haplotype analysis,the most significantly different haplotype was H4:ACGAAC,with 13.1％ frequency in the GPP group but only 3.4％ in the control group (OR =4.16,P =4.459 × 10-7).However,no significant difference in the allele frequencies was found between the PPP group and control group for each of the six SNPs (P ＞ 0.05).Conclusions:Polymorphisms in TNIP1 are associated with GPP in Chinese Han population.However,no association with PPP was found.These findings suggest that TNIP1 might be a susceptibility gene for GPP.

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the σ factor/anti-σ factorprotein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of σ factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems.

Full Text Available Large-scale data sets of protein-protein interactions (PPIs are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.e., the interacting regions, IRs suggested to correspond to functional and structural domains. Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs, including 12 transcription-related proteins. The core data set (966 IRs; 943 PPIs displays a verification rate of 70%. Analysis of the IR data set revealed the existence of IRs that interact with multiple partners. Furthermore, these IRs were preferentially associated with intrinsic disorder. This finding supports the hypothesis that intrinsically disordered regions play a major role in the dynamics and diversity of TF networks through their ability to structurally adapt to and bind with multiple partners. Accordingly, this domain-based interaction resource represents an important step in refining protein interactions and networks at the domain level and in associating network analysis with biological structure and function.

The insulin-like growth factor binding protein (Igfbp) family consists of six members designated Igfbp1-6. Igfbps are involved in many vital biological functions. They physically interact with IGFs (IGF1 and IGF2) and act as carriers, thereby protecting IGFs from proteolytic degradation. Thus, they function as modulators of IGF activity. Furthermore, Igfbps have been reported to have IGF-independent activities. They interact with other proteins, including cell surface proteins, extra-cellular matrix proteins, and potentially intracellular molecules. In Xenopus tropicalis (X. tropicalis), only four igfbp genes (igfbp1, igfbp2, igfbp4, and igfbp5) have been identified, and their expression is not well characterized. We report that X. tropicalis genome lacks the igfbp3 and igfbp6 genes based on synteny analyses. We also examined the spatio-temporal expression patterns of igfbp genes in early X. tropicalis development. Expression analyses indicated that they are differentially expressed during early development. Each igfbp gene showed a characteristic spatial expression pattern. Except for igfbp5, they demonstrated overlapping expression in the pronephros. The Xenopus pronephros is composed of four domains (i.e., the proximal tubule, intermediate tubule, distal tubule, and connecting tubule). Our results showed that at least two igfbp genes are co-expressed in all pronephric domains, suggesting that redundant functions of igfbp genes are required in early pronephric kidney development.

Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx) fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method. Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni(2+) affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF.

Full Text Available Intravenous nanoemulsions have been on the market for parenteral nutrition since the 1950s; meanwhile, they have also been used successfully for IV drug delivery. To be well tolerable, the emulsions should avoid uptake by the MPS cells of the body; for drug delivery, they should be target-specific. The organ distribution is determined by the proteins adsorbing them after injection from the blood (protein adsorption pattern, typically analyzed by two-dimensional polyacrylamide gel electrophoresis, 2-D PAGE. The article reviews the 2-D PAGE method, the analytical problems to be faced and the knowledge available on how the composition of emulsions affects the protein adsorption patterns, e.g., the composition of the oil phase, stabilizer layer and drug incorporation into the interface or oil core. Data were re-evaluated and compared, and the implications for the in vivo distribution are discussed. Major results are that the interfacial composition of the stabilizer layer is the main determining factor and that this composition can be modulated by simple processes. Drug incorporation affects the pattern depending on the localization of the drug (oil core versus interface. The data situation regarding in vivo effects is very limited; mainly, it has to be referred to in the in vivo data of polymeric nanoparticles. As a conclusion, determination of the protein adsorption patterns can accelerate IV nanoemulsion formulation development regarding optimized organ distribution and related pharmacokinetics.

Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.

of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha......The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... noticed that components of 136 and 97 kDa bound to a peptide from this region of the receptor in a phosphorylation-independent manner. These components have now been purified and identified as alpha- and beta'-coatomer proteins (COPs), respectively. COPs are a family of proteins involved in the regulation...

Meningiomas are the second most common primary intracranial tumors in adults. Although meningiomas are mostly benign, more than 50% of patients with meningioma develop peritumoral brain edema (PTBE), which may be fatal because of increased intracranial pressure. Vascular endothelial growth factor....... Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression...... in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p

Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.

A wide variety of factors can influence the development, production and introduction of high-quality protein foods in a given country. Such factors can be grouped in three main areas: I. Factors depending upon the country itself. II. Factors related with the identity of the food and III. Factors inherent to the consumer. The role of the food industry and of the government are discussed in area I, and such aspects as improvement of staples, availability of raw materials, health programs and energy crisis are briefly commented. Area II covers product identity in relation to used ingredients. Nutritional quality and requirements as well as the danger of increasing the price of the product after being in the market are briefly discussed. The consumer's attitude, preferences and personal reactions towards the presentation of the food are covered in area III. Also marketing approach, promotion, labels and possible influence of the name are discussed. The launching of "incaparina" in Venezuela in 1964 and the reasons for its failure are commented from the different points of view covered in the above sections.

Asp residues are significantly under represented in {beta}-sheet regions of proteins, especially in the middle of {beta}-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a {beta}-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V{sub L}) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a {beta}-strand, and that of Q89D, located in the middle of a {beta}-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V{sub L}-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V{sub L}-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V{sub L}-Len domain. The structures of mutants V{sub L}-Len Q38D and V{sub L}-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these QD mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a {beta}-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a {beta}-strand residue to Asp could prevent the expression of the

Full Text Available BACKGROUND: Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2 is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2 from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. CONCLUSION: Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths.

Multidrug resistance (MDR) is one of the most important causes of chemotherapy failure and carcinoma recurrence.But the roles of the MDR-associated protein MRP1 in MDR remain poorly understood.Vascular endothelial growth factor (VEGF),one of the most active and specific vascular growth factors,plays a significant role in proliferation,differentiation,and metastasis of cancers.To explore the effect of VEGF on the expression of MRP1,we used recombinant human VEGF to stimulate K562 and BGC-823 cell lines.Quantitative real-time polymerase chain reaction and western blot analysis showed that the expression of MRP1 at both mRNA and protein levels was increased.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results also showed that VEGF significantly enhanced the ICs0 of the cells treated with adriamycin.To explore the underlying regulatory mechanisms,we constructed MRP1 promoter and the luciferase reporter gene recombinant vector.The luciferase reporter gene assay showed that the activity of the MRP1 promoter was markedly increased by VEGF stimulation,while LY294002,an inhibitor of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway,reduced this effect.Transcription factor specificity protein 1 (SP1) binding site mutation partially blocked the up-regulation of MRP1 promoter activity by VEGF.In summary,our results demonstrated that VEGF enhanced the expression of MRP1,and the PI3K/Akt signaling pathway and SP1 may be involved in this modulation.

Full Text Available Yingqian Lv, Shan Zhao, Jinzhu Han, Likang Zheng, Zixin Yang, Li Zhao Department of Oncology, The Second Hospital, Hebei Medical University, Shijiazhuang, Hebei Province, People’s Republic of China Abstract: Multidrug resistance is the major cause of chemotherapy failure in many solid tumors, including colon cancer. Hypoxic environment is a feature for all solid tumors and is important for the development of tumor resistance to chemotherapy. Hypoxia-inducible factor (HIF-1α is the key transcription factor that mediates cellular response to hypoxia. HIF-1α has been shown to play an important role in tumor resistance; however, the mechanism is still not fully understood. Here, we found that HIF-1α and the drug resistance-associated gene multidrug resistance associated protein 1 (MRP1 were induced by treatment of colon cancer cells with the hypoxia-mimetic agent cobalt chloride. Inhibition of HIF-1α by RNA interference and dominant-negative protein can significantly reduce the induction of MRP1 by hypoxia. Bioinformatics analysis showed that a hypoxia response element is located at -378 to -373 bp upstream of the transcription start site of MRP1 gene. Luciferase reporter assay combined with mutation analysis confirmed that this element is essential for hypoxia-mediated activation of MRP gene. Furthermore, RNA interference revealed that HIF-1α is necessary for this hypoxia-driven activation of MRP1 promoter. Importantly, chromatin immunoprecipitation analysis demonstrated that HIF-1α could directly bind to this HRE site in vivo. Together, these data suggest that MRP1 is a downstream target gene of HIF-1α, which provides a potential novel mechanism for HIF-1α-mediated drug resistance in colon cancer and maybe other solid tumors as well. Keywords: hypoxia, hypoxia-inducible factor-1α, multidrug resistance associated protein, transcriptional regulation, chemotherapy tolerance

In 2010-2012, the North American Specialized Coagulation Laboratory Association (NASCOLA) distributed 12 proficiency testing challenges to evaluate laboratory testing for protein S (PS). Results were analysed to assess the performance of PS activity, PS free antigen, and PS total antigen testing. Statistical analysis was performed on the numeric results and qualitative classification submitted for each method. There were 2,106 total results: 716 results from PS activity assays, 833 results from PS free antigen assays, and 557 results from PS total antigen assays. The three assay types performed well in the classification of five normal samples and nine abnormal samples, although certain PS activity methods were more likely to classify normal samples as abnormal and one PS total antigen assay was more likely to classify abnormal samples as normal. PS activity methods were affected by interfering substances such as heterozygous or homozygous factor V Leiden mutation (underestimation) and the anticoagulant drug rivaroxaban (overestimation). In conclusion, NASCOLA laboratories using a variety of PS assays performed well in the classification of clearly normal and abnormal samples. Laboratories performing PS activity assays should be aware of potential interferences in samples positive for FV Leiden or containing certain anticoagulant medications.

Globular denatured proteins have structural properties similar to those of random coils. Experiments on denatured proteins have shown that when the temperature is increased thermal compaction may take place, resulting in a reduction of their radius of gyration Rg to range between 5% and 35% of its initial value. This phenomenon has been attributed to various causes, namely entropic, hydrophobic, and structural factors. The intrinsically disordered protein tau, which helps in nucleating and stabilizing microtubules in the axons of the neurons, also undergoes a relevant compaction process: when its temperature is increased from 293 K to 333 K its gyration radius decreases by 18%. We have performed an atomistic simulation of this molecule, at the lowest and highest temperatures of the mentioned interval, using both standard molecular dynamics and metadynamics, in parallel with small-angle X-ray scattering experiments. Using the fit of the experimental data and a genetic algorithm to select the most probable configurations among those produced in both atomistic simulations (standard MD and metadynamics), we were able to compute relevant changes, related to the temperature increase, in the average angles between residues, in the transient secondary structures, in the solvent accessible surface area, and in the number of intramolecular H-bonds. The analysis of the data showed how to decompose the compaction phenomenon into three contributions. An estimate of the entropic contribution to the compaction was obtained using the changes in the mean values of the angles between contiguous residues. The computation of the solvent accessible surface at the two temperatures allowed an estimation of the second factor contributing to the compaction, namely the increase in the hydrophobic interaction. We also measured the change in the average number of residues temporarily being in α-helices, 3-helices, PP II helices, β-sheets and β-turns. Those changes in the secondary

Full Text Available Abstract Background Bergström needle biopsy is widely used to sample skeletal muscle in order to study cell signaling directly in human tissue. Consequences of the biopsy protocol design on muscle protein quantity and quality remain unclear. The aim of the present study was to assess the impact of different events surrounding biopsy protocol on the stability of the Western blot signal of eukaryotic translation initiation factor4Ebindingprotein 1 (4E-BP1, Akt, glycogen synthase kinase-3β (GSK-3β, muscle RING finger protein 1 (MuRF1 and p70 S6 kinase (p70 S6K. Six healthy subjects underwent four biopsies of the vastus lateralis, distributed into two distinct visits spaced by 48 hrs. At visit 1, a basal biopsy in the right leg was performed in the morning (R1 followed by a second in the left leg in the afternoon (AF. At visit 2, a second basal biopsy (R2 was collected from the right leg. Low intensity mobilization (3 × 20 right leg extensions was performed and a final biopsy (Mob was collected using the same incision site as R2. Results Akt and p70 S6K phosphorylation levels were increased by 83% when AF biopsy was compared to R1. Mob condition induced important phosphorylation of p70 S6K when compared to R2. Comparison of R1 and R2 biopsies revealed a relative stability of the signal for both total and phosphorylated proteins. Conclusions This study highlights the importance to standardize muscle biopsy protocols in order to minimize the method-induced variation when analyzing Western blot signals.

Full Text Available Objective To explore the correlations of ideal cardiovascular health behaviors and factors with the level of high sensitivity C-reactive protein (hsCRP in diabetic population. Methods A cross-sectional method was used in present study. Seven thousand eight hundred and sixty-nine workers with diabetes were recruited who participated in the 2006-2007 Kailuan health examination without history of cerebral and myocardial infarction and without data incompleted. All the information was obtained from the unified questionnaire and measurement of blood biochemistry. Multivariate logistic regression analysis was used to analyze the effects of ideal cardiovascular health behaviors and factors on the level of hsCRP. Results The hsCRP concentrations decreased with the increasing numbers of ideal cardiovascular health behaviors and factors, the medians of hsCRP concentrations were 1.16mg/L, 1.11mg/L, 0.90mg/L and 0.76mg/L in 0-1, 2, 3 and 4-6 ideal cardiovascular health groups, respectively (P<0.01. Multivariate logistic regression analysis showed that, after adjustment by age, gender, triglyceride, low density lipoprotein cholesterol (LDL-C and high density lipoprotein cholesterol (HDL-C, the risk of hsCRP level exceeding 3mg/L in the subjects having 3 and 4-6 ideal cardiovascular health behaviors and factors was 0.60 times (95%CI 0.50-0.72 and 0.45 times (95%CI 0.34-0.61, respectively, than those having 0-1 ideal cardiovascular health behaviors and factors. Conclusion The hsCRP concentrations decreased with increasing numbers of ideal cardiovascular health behaviors and factors; the risk of hsCRP exceeding 3mg/L can be reduced by ideal cardiovascular health behaviors and factors. DOI: 10.11855/j.issn.0577-7402.2013.10.013

of type-2 diabetes and atherosclerotic cardiovascular diseases. Overweight children have higher concentrations of the metabolic syndrome risk factors than normal weight children and the pathological condition underlying cardiovascular diseases, called atherosclerosis, seems to start in childhood. A well...... in the adolescents. The analyses showed that increased physical activity was related to an improved arterial function whereas central adiposity and a high protein intake were related to increased arterial stiffness. In the intervention study, the adolescents with habitual low milk intakes were randomized to drink 1L...... factors than children having a high milk intake. The aim of the intervention study was to examine whether it is beneficial for overweight adolescents with a habitual low milk intake to increase the consumption of low fat milk and whether a potential beneficial effect is caused by whey or casein. The data...

Tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) have proved to be important in rheumatoid arthritis (RA) because the outcome of RA has greatly improved with the recent availability of biologics targeting them. It is well accepted that these cytokines are involved in the activation of the nuclear factor-κB (NF-κB) signaling pathway, but our understanding of the dependency of these pro-inflammatory cytokines and the link between them in RA is currently limited. Recently, we and others proved the importance of TNFα-induced protein (TNFAIP), due to the spontaneous development of arthritis in deficient animals that are dependent on IL-6. To date, nine TNFAIPs have been identified, and TNFAIP3 and TNFAIP9 were found to be clearly associated with mouse and human arthritis. In this review, we compare and discuss recent TNFAIP topics, especially focusing on TNFAIP3 and TNFAIP9 in autoimmune arthritis in mice and humans.

Full Text Available BACKGROUND: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP which interact with complement regulator factor H (CFH and factor H-like protein 1 (FHL1 or factor H-related protein 1 (CFHR1. In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement. CONCLUSIONS/SIGNIFICANCE: In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi.

Preadipocyte factor-1 (Pref-1)/delta-like protein/fetal antigen-1 (FA1) is a member of the epidermal growth factor-like family. It is widely expressed in embryonic tissues, whereas in adults it is confined to the adrenal gland, the anterior pituitary, the endocrine pancreas, the testis and the ov......Preadipocyte factor-1 (Pref-1)/delta-like protein/fetal antigen-1 (FA1) is a member of the epidermal growth factor-like family. It is widely expressed in embryonic tissues, whereas in adults it is confined to the adrenal gland, the anterior pituitary, the endocrine pancreas, the testis...

Polymeric tissue engineering scaffolds prepared by conventional techniques like salt leaching and phase separation are greatly limited by their poor biomolecule-delivery abilities. Conventional methods of incorporation of various growth factors, proteins, and/or peptides on or in scaffold materials via different crosslinking and conjugation techniques are often tedious and may affect scaffold's physical, chemical, and mechanical properties. To overcome such deficiencies, a novel two-step porous scaffold fabrication procedure has been created in which bovine serum albumin microbubbles (henceforth MB) were used as porogen and growth factor carriers. Polymer solution mixed with MB was phase separated and then lyophilized to create porous scaffold. MB scaffold triggered substantially lesser inflammatory responses than salt-leached and conventional phase-separated scaffolds in vivo. Most importantly, the same technique was used to produce insulin-like growth factor-1 (IGF-1)-eluting porous scaffolds, simply by incorporating IGF-1-loaded MB (MB-IGF-1) with polymer solution before phase separation. In vitro such MB-IGF-1 scaffolds were able to promote cell growth to a much greater extent than scaffold soaked in IGF-1, confirming the bioactivity of the released IGF-1. Further, such MB-IGF-1 scaffolds elicited IGF-1-specific collagen production in the surrounding tissue in vivo. This novel growth factor-eluting scaffold fabrication procedure can be used to deliver a range of single or combination of bioactive biomolecules to substantially promote cell growth and function in degradable scaffold.

Full Text Available Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs. Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I.

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulation factors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulation factors via their γ-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulation factors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulation factors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10β1-β3 ((23)MEMKN ISALK HGKNN LRFKF RGIKI QVL(60)) bound to immobilized prothrombin, and mutants that contained SSL10β10-β12 ((174)SFYNL DLRSK LKFKY MGEVI ESKQI KDIEV NLK(207)) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to prothrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants.

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.

The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among th...

The alternative sigma factor E (σ(E)) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ(E)-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ(E) may indirectly participate in post-transcriptional regulation. In this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ(E) in Salmonella. Samples were analyzed from wild-type and isogenic rpoE mutant Salmonella cultivated in three different conditions: nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of the observed proteome was regulated by σ(E) combining all three conditions. In different growth conditions, σ(E) affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ(E) and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ(E)-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ(E)-mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region,three tandem Kunitz-type domains,and a positively charged carboxyterminal tail.Human TFPI-2 (hTFPI-2) inhibits a broad spectrum of serine proteinases (including trypsin,plasmin,plasma kallikrein,XIa,and chymotrypsin) almost exclusively via its first Kunitz-type domain,and potentially plays an important role in the regulation of extracellular matrix digestion and remodeling [1].Reduced TFPI-2 synthesis has been related to numerous pathophysiological processes such as inflammation,angiogenesis,atherosclerosis [2,3],retinal degeneration,and tumor growth/metastasis [4-6].It has been suggested that TFPI-2 is a tumor suppressor gene in some cancers [7,8].However,the specific physiological functions of hTFPI-2 in humans are unclear,particularly its interactions with other proteins.To better understand the physiological function of hTFPI-2,we used yeast two-hybrid system screening and bioinformatics analysis to identify its interacting proteins and confirm its interactions with nuclear protein RASSF1C using confocal microscopy and co-immunoprecipitation.

Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.

Sequence similarity has given rise to the proposal that IF-2, EF-G, and EF-Tu are related through a common ancestor. We evaluate this proposition and whether the relationship can be extended to other factors of protein synthesis. Analysis of amino acid sequence similarity gives statistical support for an evolutionary affiliation among IF-1, IF-2, IF-3, EF-Tu, EF-Ts, and EF-G and suggests further that this association is a result of gene duplication/fusion events. In support of this mechanism, the three-dimensional structures of IF-3, EF-Tu, and EF-G display a predictable domain structure and overall conformational similarity. The model that we propose consists of three consecutives duplication/fusion events which would have taken place before the divergence of the three superkingdoms: eubacteria, archaea, and eukaryotes. The root of this protein superfamily tree would be an ancestor of the modern IF-1 gene sequence. The repeated fundamental motif of this protein superfamily is a small RNA binding domain composed of two alpha-helices packed along side of an antiparallel beta-sheet.

TEAD (TEA/ATTS domain) transcription factors are the most distal effectors of the Hippo pathway. YAP (Yes-associated protein) is a coactivator protein which, upon binding to TEAD proteins, stimulates their transcriptional activity. Since the Hippo pathway is deregulated in various cancers, designing inhibitors of the YAP:TEAD interaction is an attractive therapeutic strategy for oncology. Understanding the molecular events that take place at the YAP:TEAD interface is therefore important not only to devise drug discovery approaches, but also to gain knowledge on TEAD regulation. In this report, combining single site-directed mutagenesis and double mutant analyses, we conduct a detailed analysis on the role of several residues located at the YAP:TEAD interface. Our results provide quantitative understanding of the interactions taking place at the YAP:TEAD interface and give insights into the formation of the YAP:TEAD complex and more particularly on the interaction between TEAD and the Ω-loop found in YAP.

Numerous studies have confirmed that oligodendrocyte transcription factor 1 (Olig-1) is vital for myelin repair. However, the effects of hypoxia and ischemia on Olig-1 expression remain unknown.In this study, Olig-1 mRNA and protein expressions were analyzed by in situ hybridization and immunohistochemistry, to determine the expression profile of Olig-1 in rat brain slices exposed to hypoxia and ischemia. Brains were obtained from 2-day-old Sprague-Dawley rats, and sections were randomly assigned to control and hypoxia/ischemia groups. Hematoxylin-eosin staining revealed karyorrhexis and karyopyknosis in cells from the hypoxia/ischemia group. Under electron microscopy, mitochondria swelling and neuropil edema were observed in the hypoxia/ischemia group. Olig-1 mRNA and protein expressions were increased at 1 day after hypoxia and ischemia treatment. These results suggest that in situ hybridization and immunohistochemistry could be used simultaneously to detect mRNA and protein expression in brain slices.

Full Text Available Familial amyloidotic polyneuropathy (FAP is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR. This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer's and Parkinson's. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.

Full Text Available Insulin-like growth factor binding protein 7 (IGFBP-7 is the only member of the IGFBP superfamily that binds strongly to insulin, suggesting that IGFBP-7 may have different functions from other IGFBPs. Unlike other IGFBPs, the expression and functions of IGFBP-7 in glioma tumors have not been reported. Using cDNA microarray analysis, we found that expression of IGFBP-7 correlated with the grade of glioma tumors and the overall patient survival. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. We used RNAi to examine the role of IGFBP-7 in glioma cells, inhibiting IGFBP-7 expression by short interfering RNA transfection. Cell proliferation was suppressed after IGFBP-7 expression was inhibited for 5 days, and glioma cell growth was stimulated consistently by the addition of recombinant IGFBP-7 protein. Moreover, glioma cell migration was attenuated by IGFBP-7 depletion but enhanced by IGFBP-7 overexpression and addition. Overexpression of AKT1 in IGFBP-7-overxpressed cells attenuated the IGFBP-7-promoted migration and further enhanced inhibition of IGFBP-7 depletion on the migration. Phosphorylation of AKT and Erk1/2 was also inversely regulated by IGFBP-7 expression. These two factors together suggest that IGFBP-7 can regulate glioma cell migration through the AKT-ERK pathway, thereby playing an important role in glioma growth and migration.

Full Text Available Atherosclerosis is the leading cause of death in the developed world1.In Pakistan; the disease seems to follow an accelerated course with ischaemic events occurring a decade earlier than those reported worldwide2. While many risk factors of cardiovascular diseases are well established, many are still under evaluation; like homocysteine, which is now being considered an independent risk factor for cardiovascular diseases3.Homocysteine is a sulfur containing amino acid which is not our dietary constituent but is formed by metabolism of methionine, another amino acid present in our daily protein diet. In case of excess formation of homocysteine as compared to its consumption, it appears in urine if levels are too high3. Normal plasma homocysteine concentration ranges from 5 to 15μmol/litre. Of this, almost 75% is bound to proteins, especially albumin, through disulfide bond. Classification of hyperhomocysteinemia described by Kang SS 4 is followed widely.Moderate hyperhomocysteinemia (15 to 30 μmol/litreIntermediate hyperhomocysteinemia (30 to 100μmol/litreSevere hyperhomocysteinemia (>100 μmol/litre

The acquisition of regulatory proteins is a means of blood-borne pathogens to avoid destruction by the human complement. We recently showed that the gametes of the human malaria parasite Plasmodium falciparum bind factor H (FH) from the blood meal of the mosquito vector to assure successful sexual reproduction, which takes places in the mosquito midgut. While these findings provided a first glimpse of a complex mechanism used by Plasmodium to control the host immune attack, it is hitherto not known, how the pathogenic blood stages of the malaria parasite evade destruction by the human complement. We now show that the human complement system represents a severe threat for the replicating blood stages, particularly for the reinvading merozoites, with complement factor C3b accumulating on the surfaces of the intraerythrocytic schizonts as well as of free merozoites. C3b accumulation initiates terminal complement complex formation, in consequence resulting in blood stage lysis. To inactivate C3b, the parasites bind FH as well as related proteins FHL-1 and CFHR-1 to their surface, and FH binding is trypsin-resistant. Schizonts acquire FH via two contact sites, which involve CCP modules 5 and 20. Blockage of FH-mediated protection via anti-FH antibodies results in significantly impaired blood stage replication, pointing to the plasmodial complement evasion machinery as a promising malaria vaccine target.

Full Text Available Interaction of prion protein and amyloid-b oligomers has been demonstrated recently. Homozygosity at prion protein gene (PRNP codon 129 is associated with higher risk for Creutzfeldt-Jakob disease. This polymorphism has been addressed as a possible risk factor in Alzheimer disease (AD. Objective To describe the association between codon 129 polymorphisms and AD. Methods We investigated the association of codon 129 polymorphism of PRNP in 99 AD patients and 111 controls, and the association between this polymorphism and cognitive performance. Other polymorphisms of PRNP and additive effect of apolipoprotein E gene (ApoE were evaluated. Results Codon 129 genotype distribution in AD 45.5% methionine (MM, 42.2% methionine valine (MV, 12.1% valine (VV; and 39.6% MM, 50.5% MV, 9.9% VV among controls (p>0.05. There were no differences of cognitive performance concerning codon 129. Stratification according to ApoE genotype did not reveal difference between groups. Conclusion Codon 129 polymorphism is not a risk factor for AD in Brazilian patients.

Insulin-like growth factor II mRNA-binding protein 1 (IMP1) belongs to a family of RNA-binding proteins implicated in mRNA localization, turnover, and translational control. Mouse IMP1 is expressed during early development, and an increase in expression occurs around embryonic day 12.5 (E12.5). T...

The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3...

Fat-specific protein 27 (FSP27), a member of the cell death-inducing DNA fragmentation factor a-like effector (Cide) family, is highly expressed in adipose tissues and is a lipid droplet (LD)-associated protein that induces the accumulation of LDs. Using a yeast two-hybrid system to examine potentia...

Full Text Available Background: Hypoxia modifies the phenotype of tumors in a way that promotes tumor aggressiveness and resistance towards chemotherapy and radiotherapy. However, the expression and influence of hypoxia-regulated proteins on tumor biology are not well characterized in colorectal tumors. We studied the role of protein expression of hypoxia-inducible factor (HIF-1α, HIF-2α, carbonic anhydrase 9 (CA9 and glucose transporter 1 (GLUT1 in patients with colorectal adenocarcinomas. Methods: Expression of HIF-1α, HIF-2α, CA9 and GLUT1 was quantified by immunohistochemistry in 133 colorectal adenocarcinomas. The expression of hypoxia markers was correlated with clinicopathological variables and overall patient survival. Results: Expression of these hypoxia markers was detected in the epithelial compartment of the tumor cells as well as in tumor-associated stromal cells. Although tumor cells frequently showed expression of one or more of the investigated hypoxia markers, no correlation among these markers or with clinical response was found. However, within the tumor stroma, positive correlations between the hypoxia markers HIF-2α, CA9 and GLUT1 were observed. Furthermore expression of HIF-2α and CA9 in tumor-associated stroma were both associated with a significantly reduced overall survival. In the Cox proportional hazard model, stromal HIF-2α expression was an independent prognostic factor for survival. Conclusion: These observations show, that expression of hypoxia regulated proteins in tumor-associated stromal cells, as opposed to their expression in epithelial tumor cells, is associated with poor outcome in colorectal cancer. This study suggests that tumor hypoxia may influence tumor-associated stromal cells in a way that ultimately contributes to patient prognosis.

each. Fasting blood samples were collected before and after the dietary regimen and analysed for triglycerides, FVII:C, and protein concentrations of FVII, FII, FX, protein C, CRP, albumin, fibrinogen, and F1 + 2. FVII:C was significantly reduced on diet S compared with diet ML. This was accompanied...... by a decrease in FVII protein, F1 + 2 and the vitamin K-dependent proteins FII, FX, and protein C. In contrast, no changes were observed in triglycerides, FVII:C/FVII:Ag, albumin and CRP. Fibrinogen was increased on diet S compared with diet ML. Our findings suggest that the change in fasting FVII:C was part...... of a general change in concentrations of vitamin K-dependent proteins. Udgivelsesdato: 1995-Feb...

Full Text Available Pax transactivation domain interacting protein (PTIP associated protein 1, PA1, was a newly found protein participating in the modulation of transactivity of nuclear receptor super family members such as estrogen receptor (ER, androgen receptor (AR and glucocorticoid receptor (GR. Breast cancer is one of the most life threatening diseases for women and has tight association with estrogen and ER. This study was performed to understand the function of PA1 in breast cancer. The expression of PA1 had been evaluated in a total of 344 primary invasive breast cancer samples and examined the relationship with clinical output, relapse free survival (RFS, breast cancer-specific survival (BCSS. PA1 expression was observed in both nucleus and cytoplasm, however, appeared mainly in nuclear. PA1 nuclear expression was correlated with postmenopausal (P = 0.0097, smaller tumor size (P = 0.0025, negative Ki67 (P = 0.02, positive AR (P = 0.049 and positive ERβ (P = 0.0020. Kaplan-Meier analysis demonstrated PA1 nuclear positive cases seemed to have a longer survival than negative ones for RFS (P = 0.023 but not for BCSS (P = 0.23. In the Cox hazards model, PA1 nuclear protein expression proved to be a significant prognostic univariate parameter for RFS (P = 0.03, but not for BCSS (P = 0.20. In addition, for those patients without lymphnode metastasis PA1 was found to be an independent prognostic factor for RFS (P = 0.025, which was verified by univariate and multivariate analyses. These investigations suggested PA1 expression could be a potential prognostic indicator for RFS in breast cancer.

Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

Full Text Available Background: Meningiomas are common slow-growing primary central nervous system tumors that arise from the meningothelial cells of the arachnoid and spinal cord. Human epidermal growth factor receptor 2 (HER2 or HER2/neu (also known as c-erbB2 is a 185-kD transmembrane glycoprotein with tyrosine kinase activity expressed in meningiomas and various other tumors. It can be used in targeted therapy for HER2/neu positive meningiomas. Aim: To correlate the expression of HER2/neu protein in meningiomas with gender, location, histological subtypes, and grade. Materials and Methods: It was 3½ years prospective (March 2010–October 2011 and retrospective (May 2008–February 2010 study of histopathologically diagnosed intracranial and intraspinal meningiomas. Clinical details of all the cases were noted from the computerized hospital information system. Immunohistochemistry for HER2/neu protein was performed along with scoring. Statistical analysis was done using Chi-square test to look for any association of HER2/neu with gender, location, grade, and various histological subtypes of meningiomas at 5% level of significance. Results: A total of 100 cases of meningiomas were found during the study period. Of which, 80 were Grade I, 18 were Grade II, and 2 were Grade III meningiomas as per the World Health Organization 2007 criteria. The female-male ratio was 1.9:1 and the mean age was 47.8 years. HER2/neu protein was expressed in 75% of Grade I and 72.2% of Grade II and none of Grade III meningiomas. About 72.7% brain invasive meningiomas showed HER2/neu immunopositivity. Conclusion: HER2/neu protein was expressed in 73% of meningiomas. Statistically significant difference of HER2/neu expression was not seen between females and males of Grade I and Grade II/III meningiomas, intracranial and spinal tumors, Grade I and Grade II/III cases, and various histological subtypes of meningiomas.

Epidermal growth factor (EGF) has been localized in human salivary and Brunner's glands and found to stimulate the proliferation of gastrointestinal and pancreatic tissues in animals, but little is known about EGF in human pancreas. This study was designed to determine the distribution and release of EGF in the pancreas and to assess the secretion of EGF and protein by the transplanted human pancreas. The peroxidase antiperoxidase (PAP) immunocytochemical method with anti-hEGF showed that EGF was restricted mainly to the excretory cells lining pancreatic ducts. The EGF immunoreactivity in the pancreatic tissue averaged about 15 +/- 0.5 micrograms/g of tissue wt. The concentration and output of EGF in the pancreatic juice were, respectively, about 3.4 +/- 0.7 ng/mL and 68 + 12 ng/h in basal secretion collected from the whole pancreatic transplant. A significant increase in EGF release from this transplant started about 2 h after its reperfusion and was accompanied by a parallel increase in protein output. Injection of iv secretion (1 U/kg) resulted in a transient rise in EGF output, probably as a result of washout by increased vol flow, whereas HCCK (1 U/kg) caused more prolonged release of EGF accompanied by a marked stimulation of protein secretion. Ingestion of a mixed meal caused an immediate and sustained increment in EGF output, and protein output showed a more protracted increase, reaching its peak in the second postprandial hour. Fractionation of an extract of pancreatic juice on G-5O Sephadex superfine column revealed that EGF immunoreactivity emerged as a major peak in the same position as authentic human EGF (hEGF).(ABSTRACT TRUNCATED AT 250 WORDS)

A single protein of Mr 17,000-19,000 and pI approximately equal to 5.1, found in all animal cells we have studied to date, undergoes post-translational modification in growing cells to form the unusual amino acid hypusine. Because of the association of this modification with the increasing rate of protein synthesis during lymphocyte growth stimulation, its subcellular distribution, and its widespread occurrence and structural conservation among animal cells, we considered the possibility that this protein might be a translation initiation factor. Purified rabbit reticulocyte factors (eukaryote initiation factors) eIF-4C and eIF-4D were chosen for study because of their Mr (17,000-19,000) and acidic pI. The hypusine-containing protein and purified eIF-4D showed identity of electrophoretic mobility in both isoelectric focusing and NaDodSO4/polyacrylamide gel electrophoresis dimensions, while eIF-4C was clearly nonidentical. Purified eIF-4D contained approximately 1 mol of hypusine per mol of protein. Since only one protein has thus far been observed to contain hypusine, we conclude that eIF-4D is the hypusine-containing protein. On the basis of relative synthesis among lymphocyte proteins and detection by Coomassie blue staining, we also conclude that eIF-4D is a major cell protein. It is possible that the activity of this factor is modulated by It is possible that the activity of this factor is modulated by post-translational hypusine formation, which may play a role in regulation of protein synthesis during lymphocyte growth stimulation.

Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans. PMID:27138431

Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.

Full Text Available Jatropha curcas is a multipurpose tree, which has potential as an alternative source for biodiesel. All of its parts can also be used for human food, animal feed, fertilizer, fuel and traditional medicine. J. curcas seed cake is a low-value by-product obtained from biodiesel production. The seed cake, however, has a high amount of protein, with the presence of a main toxic compound: phorbol esters as well as anti-nutritional factors: trypsin inhibitors, phytic acid, lectin and saponin. The objective of this work was to detoxify J. curcas seed cake and study the toxin, anti-nutritional factors and also functional properties of the protein isolated from the detoxified seed cake. The yield of protein isolate was approximately 70.9%. The protein isolate was obtained without a detectable level of phorbol esters. The solubility of the protein isolate was maximal at pH 12.0 and minimal at pH 4.0. The water and oil binding capacities of the protein isolate were 1.76 g water/g protein and 1.07 mL oil/g protein, respectively. The foam capacity and stability, including emulsion activity and stability of protein isolate, had higher values in a range of basic pHs, while foam and emulsion stabilities decreased with increasing time. The results suggest that the detoxified J. curcas seed cake has potential to be exploited as a novel source of functional protein for food applications.

Alzheimer's disease (AD) has long been viewed as a pathology that must be caused either by aberrant amyloid-β protein precursor (AβPP) processing, dysfunctional tau protein processing, or a combination of these two factors. This is a reasonable assumption because amyloid-β peptide (Aβ) accumulation and tau hyperphosphorylation are the defining histological features in AD, and because AβPP and tau mutations can cause AD in humans or AD-like features in animal models. Nonetheless, other protein players are emerging that one can argue are significant etiological players in subsets of AD and potentially novel, druggable targets. In particular, the microtubule-associated protein CRMP2 (collapsin response mediator protein-2) bears striking analogies to tau and is similarly relevant to AD. Like tau, CRMP2 dynamically regulates microtubule stability; it is acted upon by the same kinases; collects similarly in neurofibrillary tangles (NFTs); and when sequestered in NFTs, complexes with critical synapse-stabilizing factors. Additionally, CRMP2 is becoming recognized as an important adaptor protein involved in vesicle trafficking, amyloidogenesis and autophagy, in ways that tau is not. This review systematically compares the biology of CRMP2 to that of tau in the context of AD and explores the hypothesis that CRMP2 is an etiologically significant protein in AD and participates in pathways that can be rationally engaged for therapeutic benefit.

The cell population and the activity of the organizer change during the course of development. We addressed the mechanism of mouse node development via an analysis of the node/notochord enhancer (NE) of Foxa2. We first identified the core element (CE) of the enhancer, which in multimeric form drives gene expression in the node. The CE was activated in Wnt/beta-catenin-treated P19 cells with a time lag, and this activation was dependent on two separate sequence motifs within the CE. These same motifs were also required for enhancer activity in transgenic embryos. We identified the Tead family of transcription factors as binding proteins for the 3' motif. Teads and their co-factor YAP65 activated the CE in P19 cells, and binding of Tead to CE was essential for enhancer activity. Inhibition of Tead activity by repressor-modified Tead compromised NE enhancer activation and notochord development in transgenic mouse embryos. Furthermore, manipulation of Tead activity in zebrafish embryos led to altered expression of foxa2 in the embryonic shield. These results suggest that Tead activates the Foxa2 enhancer core element in the mouse node in cooperation with a second factor that binds to the 5' element, and that a similar mechanism also operates in the zebrafish shield.

Embryonic stem (ES) cells have generated enormous interest due to their capacity to self-renew and the potential for growing many different cell types in vitro. Leukemia inhibitory factor (LIF), bone morphogenetic proteins, octamer-binding protein 3 or 4, and Nanog are important factors in the maintenance of pluripotency in mouse ES cells. However, the mechanisms by which these factors regulate the pluripotency remain poorly understood. To identify other proteins involved in this process, we did a proteomic analysis of mouse ES cells that were cultured in the presence or absence of LIF. More than 100 proteins were found to be involved specifically in either the differentiation process or the maintenance of undifferentiated state. Among these, chromatin-related proteins were identified as the major proteins in nuclear extracts of undifferentiated cells. Analysis with real-time RT-PCR revealed that enrichment of these proteins in pluripotent ES cells was regulated at the transcriptional levels. These results suggest that specific chromatin-related proteins may be involved in maintaining the unique properties of pluripotent ES cells.

HIV-1 requires the cellular transcription factor CBFβ to stabilize its accessory protein Vif and promote APOBEC3G degradation. Here, we demonstrate that both isoforms of CBFβ allow for increased steady-state levels of Vif, enhanced APOBEC3G degradation, and increased viral infectivity. This conserved functional interaction enhances the steady-state levels of Vif proteins from multiple HIV-1 subtypes and is required for the degradation of all human and rhesus Vif-sensitive APOBEC3 proteins by their respective lentiviral Vif proteins.

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

Predicting crystallographic B-factors of a protein from a conventional molecular dynamics simulation is challenging, in part because the B-factors calculated through sampling the atomic positional fluctuations in a picosecond molecular dynamics simulation are unreliable, and the sampling of a longer simulation yields overly large root mean square deviations between calculated and experimental B-factors. This article reports improved B-factor prediction achieved by sampling the atomic positional fluctuations in multiple picosecond molecular dynamics simulations that use uniformly increased atomic masses by 100-fold to increase time resolution. Using the third immunoglobulin-binding domain of protein G, bovine pancreatic trypsin inhibitor, ubiquitin, and lysozyme as model systems, the B-factor root mean square deviations (mean ± standard error) of these proteins were 3.1 ± 0.2-9 ± 1 Å(2) for Cα and 7.3 ± 0.9-9.6 ± 0.2 Å(2) for Cγ, when the sampling was done for each of these proteins over 20 distinct, independent, and 50-picosecond high-mass molecular dynamics simulations with AMBER forcefield FF12MC or FF14SB. These results suggest that sampling the atomic positional fluctuations in multiple picosecond high-mass molecular dynamics simulations may be conducive to a priori prediction of crystallographic B-factors of a folded globular protein.

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).

To evaluate the performance of a Russell viper venom-based activated protein C resistance (APCR) screening test relative to DNA analysis for the factor V Leiden mutation. We evaluated the concordance between Pefakit APCR screening results and DNA analysis for 435 patients homozygous (n = 11), heterozygous (n = 310), or wild-type (n =114) for the G1691A allele. Using receiver operating characteristic analysis, we found that a cutoff of 1.89 for the APCR ratio yields a sensitivity and specificity of 99.1%. In patients with discrepant genotype-phenotype correlation, their APCR may provide a more clinically relevant result. We compared several strategies for employing reflex testing and found that performing initial APCR screening followed by confirmatory molecular analysis on a subset of cases in the borderline regions between the diagnostic groups can reduce unnecessary testing by approximately 80% without compromising diagnostic accuracy.

skimmed milk, whey, casein or water for three months. The background for the intervention is that milk is an important source of protein in the Western diet and epidemiological studies in children have shown that children drinking low amounts of milk have higher concentrations of the metabolic risk...... factors than children having a high milk intake. The aim of the intervention study was to examine whether it is beneficial for overweight adolescents with a habitual low milk intake to increase the consumption of low fat milk and whether a potential beneficial effect is caused by whey or casein. The data...... from the intervention study showed that skimmed milk, whey and casein increased the age-adjusted BMI despite that there were no changes in energy intake. Also, the whey and casein group had increased insulin secretion measured by the C-peptide concentration. The results also showed that a high intake...

Full Text Available BACKGROUND: Insulin-like growth factor binding proteins (IGFBPs are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002 FEBS lett 527: 293-297. METHODOLOGY/PRINCIPAL FINDINGS: We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6 to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells and IGFBP-5 (in C2 cells increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway. CONCLUSIONS: Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular

The unfolded-protein response (UPR), activated by sensor molecules PERK, ATF6, and IRE1 to resolve endoplasmic reticulum (ER) stress, has emerged as a key target for host cells and viruses to control the infection outcomes. The UPR regulates ER protein folding, controls cell fate upon ER stress, and plays an important role in innate immunity. We and others have shown that human cytomegalovirus (HCMV) modulates the UPR. We show here that murine CMV (MCMV), the widely used CMV model for small animal infection, regulated the UPR in a manner similar to that of HCMV. This modulatory ability was triggered by virion entry and enhanced by viral immediate-early and early gene expression. Thus, while vulnerable at early times, MCMV became resistant to exogenous ER stress at late times of infection. MCMV activated the PERK-ATF4 pathway but only induced a subset of representative ATF4 targets at levels somewhat lower than those by the ER stress inducer tunicamycin. Moreover, MCMV induced ER chaperone Bip but actively blocked IRE1-mediated Xbp1(s) protein accumulation. ATF4 depletion severely attenuated viral growth at a low multiplicity of infection by modestly reducing viral DNA synthesis and more pronouncedly inhibiting late gene transcription. Collectively, we show that the UPR is a conserved target of CMVs and identify ATF4, a key UPR component, as a factor critical for MCMV infection. This work sets the stage for using the MCMV model to explore the role of this stress response in CMV biology, particularly during infection of the host, which is difficult to study in HCMV.

A mitochondrial specific stress response termed mitochondrial unfolded protein response (UPR(mt)) is activated in responding to disturbance of protein homeostasis in mitochondria. The activating transcription factor associated with stress-1 (designated as ATFS-1) is the key regulator of UPR(mt). To investigating the roles of ATFS-1 (LvATFS-1) in Litopenaeus vannamei mitochondrial stress remission and immunity, it's full length cDNA was cloned. The open reading frame of LvATFS-1 was 1, 557 bp in length, deducing to a 268 amino acids protein. LvATFS-1 was highly expressed in muscle, hemocytes and eyestalk. Subcellular location assays showed that N-terminal of LvATFS-1 contained a mitochondrial targeting sequence, which could directed the fused EGFP located to mitochondria. And the C-terminal of LvATFS-1, which had a nuclear localization signal, expressed in nucleus. The in vitro experiments verified that LvATFS-1 could reduced the level of intracellular reactive oxygen species (ROS). And results of real-time RT-PCR indicated that LvATFS-1 might scavenge excess ROS via ROS-eliminating genes regulation. Reporter gene assays showed that LvATFS-1 could upregulated the expression of the antimicrobial peptide genes in Drosophila Schneider 2 cells. Results of real-time RT-PCR showed that Vibrio alginolyticus or white spot syndrome virus (WSSV) infection induced the expression of LvATFS-1. And knocked-down LvATFS-1 by RNAi resulted in a higher cumulative mortality of L. vannamei upon V. alginolyticus or WSSV infection. These results suggested that LvATFS-1 not only rolled in mitochondrial specific stress responding, but also important for L. vannamei immunologic defence.

The G-protein-mediated Rho guanine nucleotide exchange factor (GEF)-Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein-regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a ∼80 nM K(d) and suppresses LARG catalyzed RhoA activation dose dependently. It is active in blocking the interaction of LARG and related G-protein-coupled Rho GEFs with RhoA without a detectable effect on other DBL family Rho GEFs, Rho effectors, or a RhoGAP. In cells, Y16 selectively inhibits serum-induced RhoA activity and RhoA-mediated signaling, effects that can be rescued by a constitutively active RhoA or ROCK mutant. By suppressing RhoA activity, Y16 inhibits mammary sphere formation of MCF7 breast cancer cells but does not affect the nontransforming MCF10A cells. Significantly, Y16 works synergistically with Rhosin/G04, a Rho GTPase activation site inhibitor, in inhibiting LARG-RhoA interaction, RhoA activation, and RhoA-mediated signaling functions. Thus, our studies show that Rho GEFs can serve as selective targets of small chemicals and present a strategy of dual inhibition of the enzyme-substrate pair of GEF-RhoA at their binding interface that leads to enhanced efficacy and specificity.

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

Whether cultured human trabecular meshwork cells express transforming growth factor-β2 (TGF-β2) messenger RNA (mRNA) and protein was investigated. Total RNA of 106 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β2 messenger RNA, and the PCRproduct was verified by sequencing. Immunohistochemical staining was used to detect TGF-β2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β2 and contribute to the presence of TGF-β2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further in vestigation.

To evaluate levels of anti-carbamylated protein (anti-CarP) antibodies in rheumatoid arthritis (RA) patients and to determine their association with serological parameters and disease activity. Methods: A cross-sectional study involving 105 multiethnic RA patients (48 rheumatoid factor [RF]-positive and 57 RF-negative patients) was conducted at Hospital Universiti Sains Malaysia, Kelantan, Malaysia, from January 2015 to February 2016. Fifty healthy controls (HCs) were included. C-reactive protein (CRP), RF, anti-cyclic citrullinated peptide (anti-CCP) and anti-CarP antibodies were measured. A health assessment questionnaire (HAQ) was administered to the study participants and 28-joint Disease Activity Score (DAS28) were obtained. Results: The level of anti-CarP antibodies was significantly increased in the RA patients compared with HCs (p=0.042). The presence of anti-CarP antibodies was significantly associated with RF (p=0.019) and the HAQ (p=0.010). A significant association between the presence of anti-CarP antibodies and the DAS28 was not found (p=0.632). Conclusion: Our study provides further evidence that the level of anti-CarP antibodies is significantly elevated in RA patients.

Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

Models for exploring tyrosine nitration in proteins have been created based on 3D structural features of 20 proteins for which high resolution X-ray crystallographic or NMR data are available and for which nitration of 35 total tyrosines has been experimentally proven under oxidative stress. Factors suggested in previous work to enhance nitration were examined with quantitative structural descriptors. The role of neighboring acidic and basic residues is complex: for the majority of tyrosines that are nitrated the distance to the heteroatom of the closest charged sidechain corresponds to the distance needed for suspected nitrating species to form hydrogen bond bridges between the tyrosine and that charged amino acid. This suggests that such bridges play a very important role in tyrosine nitration. Nitration is generally hindered for tyrosines that are buried and for those tyrosines where there is insufficient space for the nitro group. For in vitro nitration, closed environments with nearby heteroatoms or unsaturated centers that can stabilize radicals are somewhat favored. Four quantitative structure-based models, depending on the conditions of nitration, have been developed for predicting site-specific tyrosine nitration. The best model, relevant for both in vitro and in vivo cases predicts 30 of 35 tyrosine nitrations (positive predictive value) and has a sensitivity of 60/71 (11 false positives). PMID:21172423

Full Text Available The initiation of premalignant lesions is associated with subtle cellular and gene expression changes. Here we describe a severe combined immunodeficient mouse xenograft model with human adult skin and compare chemical carcinogenesis and wound healing. We focus on a secreted binding protein for fibroblast growth factors (FGF-BP that enhances the activity of locally stored FGFs and is expressed at high levels in human epithelial cancers. Carcinogen treatment of murine skin induced papilloma within 6 weeks, whereas the human skin grafts displayed no obvious macroscopic alterations. Microscopic studies of the human skin, however, showed p53-positive keratinocytes in the epidermis, increased angiogenesis in the dermis of the treated skin, enhanced proliferation of keratinocytes in the basal layer, and an increase of FGF-BP protein and mRNA expression. In contrast, after surgical wounding of human skin grafts or of mouse skin, FGF-BP expression was upregulated within a few hours and returned to control levels after 2 days with wound closure. Enhanced motility of cultured keratinocytes and dermal fibroblasts by FGF-BP supports a role in wound healing. We conclude that adult human skin xenografts can be used to identify early molecular events during malignant transformation as well as transient changes during wound healing.

Leishmania tropica is one of the causative agents of cutaneous leishmaniasis. Platelet-activating factor (PAF) is a phospholipid mediator in diverse biological and pathophysiological processes. Here we show that PAF promoted a three-fold increase on ecto-protein kinase and a three-fold increase on the secreted kinase activity of L. tropica live promastigotes. When casein was added to the reaction medium, along with PAF, there was a four-fold increase on the ecto-kinase activity. When live L. tropica promastigotes were pre-incubated for 30 min in the presence of PAF-plus casein, a six-fold increase on the secreted kinase activity was observed. Also, a protein released from L. tropica promastigotes reacted with polyclonal antibodies for the mammalian CK2 alpha catalytic subunit. Furthermore, in vitro mouse macrophage infection by L. tropica was doubled when promastigotes were pre-treated for 2 h with PAF. Similar results were obtained when the interaction was performed in the presence of purified CK2 or casein. TBB and DRB, CK2 inhibitors, reversed PAF enhancement of macrophage infection by L. tropica. WEB 2086, a competitive PAF antagonist, reversed all PAF effects here described. This study shows for the first time that PAF promotes the activation of two isoforms of CK2, secreted and membrane-bound, correlating these activities to infection of mouse macrophages.

The Hippo pathway is a highly conserved potent regulator of cell growth and apoptosis including large tumor suppressor (LATS) and Yes-associated protein (YAP). LATS has been regarded as a tumor suppressor gene and YAP as either of a tumor suppressor gene or an oncogene. We investigated their expression in lung adenocarcinoma. YAP and LATS protein expression was assessed in 167 surgically resected lung adenocarcinomas and compared with clinicopathologic factors. Disease free survival and overall survival were also evaluated. YAP expression was noted in cytoplasm (48 cases; 28.7%), nuclear (34; 20.4%) and both locations (4; 2.4%). The nuclear expression was typically observed in well differentiated adenocarcinoma. LATS was expressed in cytoplasm when its signal is weak. Perinuclear expression of LATS was observed when it is strongly expressed. While cytoplasmic and nuclear YAP expressions were inversely related. In well differentiated adenocarcinoma patients, YAP nuclear expression was related with more frequent relapse. Both of nuclear YAP and LATS expression were more frequently observed in well differentiated adenocarcinoma. Furthermore, YAP expression exhibited more frequent relapse in well differentiated adenocarcinoma group. We suggest that YAP may act as an oncogene and predict poorer prognosis in well differentiated lung adenocarcinoma.

In many manufacturing and research areas, the ability to accurately monitor and characterize nanoparticles is becoming increasingly important. Nanoparticle tracking analysis is rapidly becoming a standard method for this characterization, yet several key factors in data acquisition and analysis may affect results. Nanoparticle tracking analysis is prone to user input and bias on account of a high number of parameters available, contains a limited analysis volume, and individual sample characteristics such as polydispersity or complex protein solutions may affect analysis results. This study systematically addressed these key issues. The integrated syringe pump was used to increase the sample volume analyzed. It was observed that measurements recorded under flow caused a reduction in total particle counts for both polystyrene and protein particles compared to those collected under static conditions. In addition, data for polydisperse samples tended to lose peak resolution at higher flow rates, masking distinct particle populations. Furthermore, in a bimodal particle population, a bias was seen toward the larger species within the sample. The impacts of filtration on an agitated intravenous immunoglobulin sample and operating parameters including "MINexps" and "blur" were investigated to optimize the method. Taken together, this study provides recommendations on instrument settings and sample preparations to properly characterize complex samples.

Several in vitro and in vivo studies have shown that brain-derived neurotrophic factor (BDNF) promotes survival of damaged mesencephalic dopaminergic neurons. Using a specific antibody directed against human recombinant BDNF, we studied the expression of the protein at the cellular level in the post-mortem mesencephalon of control subjects and patients with Parkinson's disease (PD). In control subjects, BDNF was expressed in all mesencephalic regions containing dopaminergic neurons, and in the substantia nigra pars compacta (SNpc) 65% of the melanized neurons expressed BDNF. In the PD SNpc, the total number of pigmented neurons containing BDNF was reduced to 9.6% of the corresponding control value. In contrast, the number of pigmented neurons non-immunoreactive for BDNF was reduced to 23.9% of the corresponding control value. This result appears to indicate that SNpc melanized neurons not expressing BDNF have a 2.5-fold greater probability of surviving than BDNF-positive melanized neurons. Furthermore, we found that in parkinsonian mesencephalon almost all dopaminergic neurons containing Lewy bodies were immunoreactive for BDNF. These findings demonstrate a reduced expression of BDNF in PD and suggest that BDNF protein expression does not protect melanized SNpc neurons from the degenerative process in this disease.

AIM: To compare the gene expression profile in a pair of HBV-infected twins.METHODS: The gene expression profile was compared in a pair of HBV-infected twins.RESULTS: The twins displayed different disease outcomes. One acquired natural immunity against HBV,whereas the other became a chronic HBV carrier. Eightyeight and forty-six genes were found to be up- or downregulated in their PBMCs, respectively. Tumor necrosis factor-alpha-induced protein 1 (TNF-αIP1) that expressed at a higher level in the HBV-immune twins was identified and four pairs of siblings with HBV immunity by RTPCR. However, upon HBV core antigen stimulation,TNF-αIP1 was downregulated in PBMCs from subjects with immunity, whereas it was slightly upregulated in HBV carriers. Bioinformatics analysis revealed a K+channel tetramerization domain in TNF-αIP1 that shares a significant homology with some human, mouse, and C elegan proteins.CONCLUSION: TNF-αIP1 may play a role in the innate immunity against HBV.

Neisseria meningitidis causes disease only in humans. An important mechanism underlying this host specificity is the ability of the organism to resist complement by recruiting the complement downregulator factor H (FH) to the bacterial surface. In previous studies, binding of FH to one of the major meningococcal FH ligands, factor H binding protein (FHbp), was reported to be specific for human FH. Here we report that sera from 23 of 73 rhesus macaques (32%) tested had high FH binding to FHbp....

Multidomain proteins constitute a large part of prokaryotic and eukaryotic proteomes and play fundamental roles in various physiological processes. However, their structural characterization is challenging because of their large size and intrinsic flexibility. We show here that motional-filtered

The establishment of the symbiosis between legume plants and rhizobial bacteria depends on the production of rhizobial lipo-chitooligosaccharidic signals (the Nod factors) that are specifically recognized by roots of the host plant. In Medicago truncatula, specific recognition of Sinorhizobium meliloti and its Nod factors requires the NFP (Nod factor perception) gene, which encodes a putative serine/threonine receptor-like kinase (RLK). The extracellular region of this protein contains three tandem lysin motifs (LysMs), a short peptide domain that is implicated in peptidoglycan or chitin binding in various bacterial or eukaryotic proteins, respectively. We report here the homology modeling of the three LysM domains of M. truncatula NFP based on the structure of a LysM domain of the Escherichia coli membrane-bound lytic murein transglycosidase D (MltD). Expression of NFP in a homologous system (M. truncatula roots) revealed that the protein is highly N-glycosylated, probably with both high-mannose and complex glycans. Surface analysis and docking calculations performed on the models of the three domains were used to predict the most favored binding modes for chitooligosaccharides and Nod factors. A convergent model can be proposed where the sulfated, O-acetylated lipo-chitooligosaccharidic Nod factor of S. meliloti binds in similar orientation to the three LysM domains of M. truncatula NFP. N-glycosylation is not expected to interfere with Nod factor binding in this orientation.

Full Text Available Factor H binding protein (fHbp is an important antigen for vaccines against meningococcal serogroup B disease. The protein binds human factor H (fH, which enables the bacteria to resist serum bactericidal activity. Little is known about the vaccine-potential of fHbp for control of meningococcal epidemics in Africa, which typically are caused by non-group B strains.We investigated genes encoding fHbp in 106 serogroup A, W-135 and X case isolates from 17 African countries. We determined complement-mediated bactericidal activity of antisera from mice immunized with recombinant fHbp vaccines, or a prototype native outer membrane vesicle (NOMV vaccine from a serogroup B mutant strain with over-expressed fHbp. Eighty-six of the isolates (81% had one of four prevalent fHbp sequence variants, ID 4/5 (serogroup A isolates, 9 (W-135, or 74 (X in variant group 1, or ID 22/23 (W-135 in variant group 2. More than one-third of serogroup A isolates and two-thirds of W-135 isolates tested had low fHbp expression while all X isolates tested had intermediate or high expression. Antisera to the recombinant fHbp vaccines were generally bactericidal only against isolates with fHbp sequence variants that closely matched the respective vaccine ID. Low fHbp expression also contributed to resistance to anti-fHbp bactericidal activity. In contrast to the recombinant vaccines, the NOMV fHbp ID 1 vaccine elicited broad anti-fHbp bactericidal activity, and the antibodies had greater ability to inhibit binding of fH to fHbp than antibodies elicited by the control recombinant fHbp ID 1 vaccine.NOMV vaccines from mutants with increased fHbp expression elicit an antibody repertoire with greater bactericidal activity than recombinant fHbp vaccines. NOMV vaccines are promising for prevention of meningococcal disease in Africa and could be used to supplement coverage conferred by a serogroup A polysaccharide-protein conjugate vaccine recently introduced in some sub

Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factorproteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts.

Full Text Available Abstract Background The repeats in toxin (Rtx are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity. Results The comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity. Conclusion In contrast to the extracellular bacteria Vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen L. pneumophila shows a rapid evolution. Changes in the rtx could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences.

Full Text Available Abstract Background Previously, we documented the presence of ovulation-inducing factor (OIF in the seminal plasma of llamas and alpacas. The purpose of the study was to define the biochemical characteristics of the molecule(s in seminal plasma responsible for inducing ovulation. Methods In Experiment 1, llama seminal plasma was centrifuged using filtration devices with nominal molecular mass cut-offs of 30, 10 and 5 kDa. Female llamas (n = 9 per group were treated i.m. with whole seminal plasma (positive control, phosphate-buffered saline (negative control, or the fraction of seminal plasma equal or higher than 30 kDa, 10 to 30 kDa, 5 to 10 kDa, or Results In Experiment 1, all llamas in the equal or higher than 30 kDa and positive control groups ovulated (9/9 in each, but none ovulated in the other groups (P Conclusions We conclude that ovulation-inducing factor (OIF in llama seminal plasma is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K, and has a molecular mass of approximately equal or higher than 30 kDa.

The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis–Menten scheme for initiation. All three initiation factors are required for maximal efficiency (kcat/KM) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations. PMID:16724118

A 37-kDa corticotropin releasing factor (CRF) binding protein (CRF-BP) was purified from human plasma by repeated affinity purification and subsequently sequenced and cloned. The human and rat CRF-BP cDNAs encode proteins of 322 amino acids with one putative signal sequence, one N-glycosylation site, and 10 conserved cysteines. Human CRF-BP binds human CRF with high affinity but has low affinity for the ovine peptide. In contrast, sheep CRF-BP binds human and ovine CRF with high affinity. The CRF-BP gene consists of seven exons and six introns and is located on chromosome 13 and loci 5q of the mouse and human genomes, respectively. CRF-BP inhibits the adrenocorticotrophic hormone (ACTH) releasing properties of CRF in vitro. CRF-BP dimerizes after binding CRF and clears the peptide from blood. This clearance mechanism protects the maternal pituitary gland from elevated plasma CRF levels found during the third trimester of human pregnancy. CRF-BP is expressed in the brains of all species so far tested but is uniquely expressed in human liver and placenta. In brain, CRF-BP is membrane associated and is predominantly expressed in the cerebral cortex and subcortical limbic structures. In some brain areas CRF-BP colocalizes with CRF and CRF receptors. The protein is also present in pituitary corticotropes, where it is under positive glucocorticoid control, and is likely to locally modulate CRF-induced ACTH secretion. The ligand requirements of the CRF receptor and the CRF-BP can be distinguished in that central human CRF fragments, such as CRF (6-33) and CRF (9-33), have high affinity for CRF-BP but low affinity for the CRF receptor. The binding protein's ability to inhibit CRF-induced ACTH secretion can be reversed by CRF (6-33) and CRF (9-33), suggesting that ligand inhibitors may have utility in elevating free CRF levels in disease states associated with decreased CRF. Thus, by controlling the amount of free CRF which activates CRF receptors, it is likely that the CRF

Full Text Available Recent studies indicate that defective activity of complement factor H (FH is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

We evaluated the state of label-free discovery proteomics focusing especially on technological contributions and contributions of naturally occurring differences in protein abundance to the intersample variability in protein abundance estimates in this highly peptide-centric technology. First, the performance of popular quantitative proteomics software, Proteome Discoverer, Scaffold, MaxQuant, and Progenesis QIP, was benchmarked using their default parameters and some modified settings. Beyond this, the intersample variability in protein abundance estimates was decomposed into variability introduced by the entire technology itself and variable protein amounts inherent to individual plants of the Arabidopsis thaliana Col-0 accession. The technical component was considerably higher than the biological intersample variability, suggesting an effect on the degree and validity of reported biological changes in protein abundance. Surprisingly, the biological variability, protein abundance estimates, and protein fold changes were recorded differently by the software used to quantify the proteins, warranting caution in the comparison of discovery proteomics results. As expected, ∼99% of the proteome was invariant in the isogenic plants in the absence of environmental factors; however, few proteins showed substantial quantitative variability. This naturally occurring variation between individual organisms can have an impact on the causality of reported protein fold changes.

Present study aimed to investigate and compare effectiveness of porous chitosan alone and in combination with insulin like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) in bone healing. Highly porous (85 ± 2%) with wide distribution of macroporous (70–900 μm) chitosan scaffolds were fabricated as bone substitutes by employing a simple liquid hardening method using 2% (w/v) chitosan suspension. IGF-1 and BMP-2 were infiltrated using vacuum infiltration with freeze drying method. Adsorption efficiency was found to be 87 ± 2 and 90 ± 2% for BMP-2 and IGF-1 respectively. After thorough material characterization (pore details, FTIR and SEM), samples were used for subsequent in vivo animal trial. Eighteen rabbit models were used to evaluate and compare control (chitosan) (group A), chitosan with IGF-1 (group B) and chitosan with BMP-2 (group C) in the repair of critical size bone defect in tibia. Radiologically, there was evidence of radiodensity in defect area from 60th day (initiated on 30th day) in groups B and C as compared to group A and attaining nearly bony density in most of the part at day 90. Histological results depicted well developed osteoblastic proliferation around haversian canal along with proliferating fibroblast, vascularization and reticular network which was more pronounced in group B followed by groups C and A. Fluorochrome labeling and SEM studies in all groups showed similar outcome. Hence, porous chitosan alone and in combination with growth factors (GFs) can be successfully used for bone defect healing with slight advantage of IGF-1 in chitosan samples. - Highlights: ► Fabrication and characterization of porous chitosan with or without IGF-1 and BMP-2 ► Highly porous growth factor loaded chitosan studied in animal subjects for 3 months ► Parameters studied: histopathology, radiology and fluorochrome labeling ► IGF-1 loaded porous chitosan found to be very effective for bone defect healing.

Gliadins, a family of wheat proteins, are central to the pathogenesis of celiac disease (CD). In addition to 'immunogenic' effects, gliadin directly affects cultured cells and intestine preparations, and produces damage in vivo, via a separate 'toxic' peptide, such as A-gliadin p31-43 (P31-43). Understanding the molecular mechanisms underlying direct non T-cell mediated effects of gliadin peptides, and assessing their potential role in promoting CD. Gliadin effects were tested on a number of cell lines and on cultured mucosa samples by evaluating cytoskeleton rearrangements, endocytosis, proliferation and apoptosis. Standard biochemical methods were used to assess prolonged epidermal growth factor receptor (EGFR) activation. Crude gliadin peptic-tryptic peptides (PTG], or P31-43 alone, fully reproduce the effects of epidermal growth factor (EGF] on actin cytosketon, cell cycle and cell proliferation of various cell lines. Inhibitor studies demonstrate the role of EGFR in the early response to gliadin exposure, pointing to activation of the EGFR pathway. Peptide P31-43 is not similar to any EGFR ligand, but can delay inactivation of the EGFR interfering with its endocytosis. Gliadin-induced delay of EGFR endocytosis in cultured intestinal biopsies, together with S-phase entry of epithelial intestinal cells, confirm a role for EGFR activation in CD. The ability of gliadin peptides to delay EGFR inactivation through interference with the endocytic pathway suggests a model where gliadin fragments amplify the effects of trace amounts of EGF, and possibly of other growth factors, by prolonging receptor activation. The results, using cultures of coeliac intestinal biopsies, highlight the role of the EGF pathway in establishing and maintaining the typical atrophic and proliferative alterations of the small intestine in CD.

The metabolic syndrome (MS) is defined by the clustering of a number of cardiovascular risk factors. The aims of the present study were to estimate the prevalence of MS in Madrid (Spain) by 2 definitions and to investigate its relationship with several sociodemographic factors and C-reactive protein (CRP) levels. This was a cross-sectional population study, and participants were 1344 subjects aged 31 to 70 years. Clinical evaluation included data on sociodemographic and cardiovascular background, physical examination, fasting glucose, triglycerides, and high-density lipoprotein cholesterol. The CRP levels were determined in a subgroup of 843 subjects. The diagnosis of MS was made according to the 2005 Adult Treatment Panel III (ATP III) and International Diabetes Federation (IDF) definitions. The age- and sex-adjusted prevalence of MS was 24.6% (95% confidence interval [CI], 22.3%-26.9%) using the ATP III definition and 30.9% (95% CI, 28.4%-33.3%) using the International Diabetes Federation definition. The overall agreement rate was 91.5% (kappa = 0.80; 95% CI, 0.76-0.83). Prevalence figures by both definitions were higher in men than in women and increased with age. Male sex, older age, low educational level, and physical inactivity were all determinants of ATP III-defined MS. The presence of MS or any of its components was associated with high CRP levels. In a logistic regression analysis, low educational level and waist circumference were the best predictors for high CRP level. The prevalence of MS in the Madrid region is one of the highest in Europe and confirms the strong Spanish regional variability in this syndrome frequency. Some sociodemographic and lifestyle factors, particularly educational level, are predictors for MS and high CRP levels.

Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation.

textabstractFetal and postnatal growth is the ultitnate result of a delicate balance between processes of proliferation, differentiation and death of cells. Proliferation and differentiation are strictly controlled by growth factors and hormones. A large number of growth factors has been characteriz

Protein kinase C (PKC) family members were examined in PC-12 rat pheochromocytoma cells to evaluate their role in the action of nerve growth factor (NGF). Immunoblot analysis of whole cell lysates using antibodies against various PKC isoforms revealed that PC-12 cells contained PKC-alpha, -delta, -epsilon and zeta. Assay of the protein kinase activity in these different anti-PKC immunoprecipitates demonstrated that NGF stimulated the kinase activity of PKC-epsilon, but not PKC-alpha, -delta a...

proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4Ebindingproteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

The TGF-β superfamily is the largest family of secreted proteins in mammals, and members of the TGF-β family are involved in most developmental and physiological processes. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), oocyte-secreted paralogs of the TGF-β superfamily, have been shown genetically to control ovarian physiology. Although previous studies found that GDF9 and BMP15 homodimers can modulate ovarian pathways in vitro, the functional species-specific significance of GDF9:BMP15 heterodimers remained unresolved. Therefore, we engineered and produced purified recombinant mouse and human GDF9 and BMP15 homodimers and GDF9:BMP15 heterodimers to compare their molecular characteristics and physiological functions. In mouse granulosa cell and cumulus cell expansion assays, mouse GDF9 and human BMP15 homodimers can up-regulate cumulus expansion-related genes (Ptx3, Has2, and Ptgs2) and promote cumulus expansion in vitro, whereas mouse BMP15 and human GDF9 homodimers are essentially inactive. However, we discovered that mouse GDF9:BMP15 heterodimer is ∼10- to 30-fold more biopotent than mouse GDF9 homodimer, and human GDF9:BMP15 heterodimer is ∼1,000- to 3,000-fold more bioactive than human BMP15 homodimer. We also demonstrate that the heterodimers require the kinase activities of ALK4/5/7 and BMPR2 to activate SMAD2/3 but unexpectedly need ALK6 as a coreceptor in the signaling complex in granulosa cells. Our findings that GDF9:BMP15 heterodimers are the most bioactive ligands in mice and humans compared with homodimers explain many puzzling genetic and physiological data generated during the last two decades and have important implications for improving female fertility in mammals. PMID:23382188

Purpose Despite most epidemiologic studies reporting that an increase in milk intake affects the growth of prostate cancer, the results of experimental studies are not consistent. In this study, we investigated the proliferation of prostate cancer cells treated with casein, the main protein in milk. Materials and Methods Prostate cancer cells (LNCaP and PC3), lung cancer cells (A459), stomach cancer cells (SNU484), breast cancer cells (MCF7), immortalized human embryonic kidney cells (HEK293), and immortalized normal prostate cells (RWPE1) were treated with either 0.1 or 1 mg/mL of α-casein and total casein extracted from bovine milk. Treatments were carried out in serum-free media for 72 hours. The proliferation of each cell line was evaluated by an 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results α-Casein and total casein did not affect the proliferations of RWPE1, HEK293, A459, SNU484, MCF7, HEK293, or RWPE1 cells. However, PC3 cells treated with 1 mg/mL of α-casein and casein showed increased proliferation (228% and 166%, respectively), and the proliferation of LNCaP cells was also enhanced by 134% and 142%, respectively. The proliferation mechanism of α-casein in PC3 and LNCaP cells did not appear to be related to the induction of Insulin-like growth factor-1 (IGF-1), since the level of IGF-1 did not change upon the supplementation of casein. Conclusions The milk protein, casein, promotes the proliferation of prostate cancer cells such as PC3 and LNCaP. PMID:25237656

Full Text Available Factor H-Binding Protein (fHbp is an outer membrane protein antigen included in two novel meningococcal group B vaccines and, as such, is an important typing target. Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical specimens only. Protocols for typing fHbp from this subset of cases have not yet been established. Here we present a nested PCR-based assay designed to amplify and sequence fHbp from non-culture clinical specimens. From analytical sensitivity experiments carried out using diluted DNA extracts, an estimated analytical sensitivity limit of 6 fg/µL of DNA (<3 genome copies/µL was calculated. The sensitivity of the assay was shown to be comparable to the ctrA-directed real-time PCR assay currently used to confirm invasive disease diagnoses from submitted clinical specimens. A panel of 96 diverse, patient-matched clinical specimen/isolate pairs from invasive disease cases was used to illustrate the breadth of strain coverage for the assay. All fHbp alleles sequenced from the isolates matched those derived from previous whole genome analyses. The first-round PCR primer binding sites are highly conserved, however an exceptional second-round PCR primer site mismatch in one validation isolate prevented amplification. In this case, amplification from the corresponding clinical specimen was achieved, suggesting that the use of a nested PCR procedure may compensate for any minor mismatches in round-two primer sites. The assay was successful at typing 91/96 (94.8% of the non-culture clinical specimens in this study and exhibits sufficient sensitivity to type fHbp from the vast majority of non-culture clinical specimens received by the Meningococcal Reference Unit, Public Health England.

AIM: To investigate the functional significance of insulin-like growth factor binding protein-5 (IGFBP-5) overexpression in pancreatic cancer (PaC).METHODS: The effects of IGFBP-5 on cell growth were assessed by stable transfection of BxPC-3 and PANC-1 cell lines and measuring cell number and DNA synthesis. Alterations in the cell cycle were assessed by flow cytometry and immunoblot analyses.Changes in cell survival and signal transduction were evaluated after mitogen activated protein kinase and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.RESULTS: After serum depr ivat ion, IGFBP-5 expression increased both cell number and DNA synthesis in BxPC-3 cells, but reduced cell number in PANC-1 cells. Consistent with this observation, cell cycle analysis of IGFBP-5-expressing cells revealed accelerated cell cycle progression in BxPC-3 and G2/M arrest of PANC-1 cells. Signal transduction analysis revealed that Akt activation was increased in BxPC-3, but reduced in PANC-1 cells that express IGFBP-5. Inhibition of PI3K with LY294002 suppressed extracellular signal-regulated kinase-1 and -2 (ERK1/2) activation in BxPC-3, but enhanced ERK1/2 activation in PANC-1 cells that express IGFBP-5. When MEK1/2 was blocked, Akt activation remained elevated in IGFBP-5 expressing PaC cells; however, inhibition of PI3K or MEK1/2 abrogated IGFBP-5-mediated cell survival.CONCLUSION: These results indicate that IGFBP-5 expression affects the cell cycle and survival signal pathways and thus it may be an important mediator of PaC cell growth.

The transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) pathways transduce extracellular signals into tissue-specific transcriptional responses. During this process, signaling effector Smad proteins translocate into the nucleus to direct changes in transcription, but how and where they localize to DNA remain important questions. We have mapped Drosophila TGF-β signaling factors Mad, dSmad2, Medea, and Schnurri genome-wide in Kc cells and find that numerous sites for these factors overlap with the architectural protein CTCF. Depletion of CTCF by RNAi results in the disappearance of a subset of Smad sites, suggesting Smad proteins localize to CTCF binding sites in a CTCF-dependent manner. Sensitive Smad binding sites are enriched at low occupancy CTCF peaks within topological domains, rather than at the physical domain boundaries where CTCF may function as an insulator. In response to Decapentaplegic, CTCF binding is not significantly altered, whereas Mad, Medea, and Schnurri are redirected from CTCF to non-CTCF binding sites. These results suggest that CTCF participates in the recruitment of Smad proteins to a subset of genomic sites and in the redistribution of these proteins in response to BMP signaling. PMID:26125535

This article is to summarize the molecular and functional analysis of the gene "suppression of tumorigenicity 13"(ST13). ST13 is in fact the gene encoding Hsp70 interacting protein (Hip), a co-factor (co-chaperone) of the 70-kDa heat shock proteins (Hsc/Hsp70). By collaborating with other positive co-factors such as Hsp40 and the Hsp70-Hsp90 organizing protein (Hop), or competing with negative co-factors such as Bc12-associated athanogen 1 (Bag1), Hip facilitates may facilitate the chaperone function of Hsc/Hsp70 in protein folding and repair, and in controlling the activity of regulatory proteins such as steroid receptors and regulators of proliferation or apoptosis. Although the nomenclature of ST13 implies a role in the suppression of tumorigenicity (ST), to date available experimental data are not sufficient to support its role in cancer development, except for the possible down-regulation of ST13 in gastric and colorectal cancers. Further investigation of this gene at the physiological level would benefit our understanding of diseases such as endocrinological disorders, cancer, and neurodegeneration commonly associated with protein misfolding.

ADP-ribosylation factor-like protein 4 (ARL4) is a GTP-binding protein which belongs to the ADP-ribosylation factorprotein (ARF) superfamily of small GTPases. ARL4 has been shown to be mainly related to the development of male germ cells and embryogenesis in mouse. To investigate the role of ARL4 i...

Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5' untranslated regions (5' UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins.......5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5' UTR-binding proteins control IGF-II biosynthesis during late mammalian development....

Full Text Available Background: Retinopathy of prematurity (ROP is the main cause of visual impairment in preterm newborn infants. Objective: This study was conducted to determine whether insulin-like growth factor binding protein -3 (IGFBP-3 is associated with proliferative ROP and has a role in pathogenesis of the disease in premature infants. Materials and Methods: A total of 71 preterm infants born at or before 32 weeks of gestation participated in this study. Studied patients consisted of 41 neonates without vaso-proliferative findings of ROP as the control group and 30 preterm infants with evidence of severe ROP in follow up eye examination as the case group. Blood samples obtained from these infants 6-8 weeks after birth and blood levels of IGFBP-3 were measured using enzyme-linked immunosorbent assay (ELISA. Results: The mean gestation age and birth weight of the studied patients were 28.2±1.6 weeks and 1120.7±197 gram in the case group and 28.4±1.6 weeks and 1189.4±454 gram in the control group (P=0.25 and P=0.44 respectively. The infants in the case group had significantly lower Apgar score at first and 5 min after birth. Insulin-like growth factor binding protein -3 (IGFBP-3 was significantly lower in the patients with proliferative ROP than the patients without ROP [592.5±472.9 vs. 995.5±422.2 ng/ml (P=0.009]. Using a cut-off point 770.45 ng/ml for the plasma IGFBP-3, we obtained a sensitivity of 65.9% and a specificity of 66.7% in the preterm infants with vasoproliferative ROP. Conclusion: Our data demonstrated that the blood levels IGFBP-3 was significantly lower in the patients with ROP and it is suspected that IGFBP-3 deficiency in the premature infants may have a pathogenetic role in proliferative ROP.

Oxaliplatin-based regimens are effective in metastasized advanced cancers. However, a major limitation to their widespread use is represented by neurotoxicity that leads to peripheral neuropathy. In this study we evaluated the roles of a proven immunotherapeutic agent [Gc-protein-derived macrophage activating factor (GcMAF)] in preventing or decreasing oxaliplatin-induced neuronal damage and in modulating microglia activation following oxaliplatin-induced damage. The effects of oxaliplatin and of a commercially available formula of GcMAF [oleic acid-GcMAF (OA-GcMAF)] were studied in human neurons (SH-SY5Y cells) and in human microglial cells (C13NJ). Cell density, morphology and viability, as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration [neuromodulin or growth associated protein-43 (Gap-43)] and markers of microglia activation [ionized calcium binding adaptor molecule 1 (Iba1) and B7-2], were determined. OA-GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability. The neuroprotective effect was accompanied by increased intracellular cAMP production, as well as by increased expression of VEGF and neuromodulin. OA-GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it increased microglial activation following oxaliplatin-induced damage, resulting in an increased expression of the markers Iba1 and B7-2 without any concomitant increase in cell number. When neurons and microglial cells were co-cultured, the presence of OA-GcMAF significantly counteracted the toxic effects of oxaliplatin. Our results demonstrate that OA-GcMAF, already used in the immunotherapy of advanced cancers, may significantly contribute to neutralizing the neurotoxicity induced by oxaliplatin, at the same time possibly concurring to an integrated anticancer effect. The association between these two powerful anticancer molecules would probably produce

Full Text Available Telomere integrity (including telomere length and capping is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography--tandem mass spectrometry (MudPIT LC-MS/MS. After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.

In mammalian cells, a single major cellular protein (eukaryotic initiation factor 4D) is post-translationally modified by the conversion of a lysine residue into the unusual amino acid hypusine. This modification was reported to occur during mitogen-stimulated growth of lymphocytes but not during quiescence, suggesting that alternative forms of eukaryotic initiation factor 4D might play a role in the regulation of cell growth perhaps through the control of protein synthesis itself (Cooper, H. L., Park, M. H., and Folk, J. E. (1982) Cell 29, 791-797). We took advantage of the drastic changes in translational specificity which occur in heat-shocked cells of Drosophila melanogaster, and of the wide variations in translation rates which occur in response to alterations of growth media in the fungus Saccharomyces cerevisiae, to investigate the relationship between the intracellular level and state of modification of the hypusine-containing protein and the rate and specificity of translation. We also studied whether the hypusine residue in this protein might be subject to further modification or reversion to lysine. Under all conditions examined, the protein was remarkably long-lived. Furthermore, the hypusine persists in this protein as hypusine, without further modification or reversion to lysine. Thus, we observe no correlation between the state of cellular translation and the persistence or reversal of this protein's modification. In addition, the data imply that neither are the state of such key cellular processes as DNA replication, RNA transcription, or carbohydrate metabolism so correlated.

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.

It is well established that protein-energy malnutrition decreases serum insulin-like growth factor (IGF)-I levels, and supplementation of 30 g of whey protein daily has been shown to increase serum IGF-I levels by 8 % after 2 years in a clinical trial. Cohort studies provide the opportunity to assess associations between dietary protein intake and IGF axis protein levels under more typical eating conditions. In the present study, we assessed the associations of circulating IGF axis protein levels (ELISA, Diagnostic Systems Laboratories) with total biomarker-calibrated protein intake, as well as with dairy product and milk intake, among postmenopausal women enrolled in the Women's Health Initiative (n 747). Analyses were carried out using multivariate linear regression models that adjusted for age, BMI, race/ethnicity, education, biomarker-calibrated energy intake, alcohol intake, smoking, physical activity and hormone therapy use. There was a positive association between milk intake and free IGF-I levels. A three-serving increase in milk intake per d (approximately 30 g of protein) was associated with an estimated average 18·6 % higher increase in free IGF-I levels (95 % CI 0·9, 39·3 %). However, total IGF-I and insulin-like growth factor-binding protein 3 (IGFBP-3) levels were not associated with milk consumption and nor were there associations between biomarker-calibrated protein intake, biomarker-calibrated energy intake, and free IGF-I, total IGF-I or IGFBP-3 levels. The findings of the present study carried out in postmenopausal women are consistent with clinical trial data suggesting a specific relationship between milk consumption and serum IGF-I levels, although in the present study this association was only statistically significant for free, but not total, IGF-I or IGFBP-3 levels.

Full Text Available Protein Energy Malnutrition (PEM continues to be a major public health problem in developing countries and affects mostly infants, young children, pregnant and lactating mothers. This study was carried on some of the factors that predispose pregnant women to PEM and hence identify groups at greater risk. A total of 1387 pregnant women (910 in the urban area and 477 in the rural areas were recruited for the study. Anthropometric indices of weight, height and Body Mass Index (BMI of the pregnant women were measured and semi structured questionnaires were used to elicit information on possible predisposing factors such as age, level of education, parity, child spacing etc. Results obtained showed that the mean weight and height of the rural pregnant women, were significantly (p<0.0001 lower than those of the urban pregnant women. The mean BMI of the rural subjects, was also significantly (p< 0.0027 lower than that of the urban subjects. Analysis of the effect of age showed that the younger age category (24 years and below had significantly (p<0.0001 lower mean BMI and higher prevalence of PEM while the effect of level of education showed significantly (p<0006 lower mean BMI and higher PEM prevalence among the less educated (no formal and primary education. Those with parity of two, one and primipara showed significantly (p<0.0175 lower mean BMI while child spacing did not have any significant effect on both mean BMI and prevalence of PEM. The implications of these findings are discussed and recommendations made on how to tackle the problem.

The evolution of the IGF binding protein (IGFBP) gene family has been difficult to resolve. Both chromosomal and serial duplications have been suggested as mechanisms for the expansion of this gene family. We have identified and annotated IGFBP sequences from a wide selection of vertebrate species as well as Branchiostoma floridae and Ciona intestinalis. By combining detailed sequence analysis with sequence-based phylogenies and chromosome information, we arrive at the following scenario: the ancestral chordate IGFBP gene underwent a local gene duplication, resulting in a gene pair adjacent to a HOX cluster. Subsequently, the gene family expanded in the two basal vertebrate tetraploidization (2R) resulting in the six IGFBP types that are presently found in placental mammals. The teleost fish ancestor underwent a third tetraploidization (3R) that further expanded the IGFBP repertoire. The five sequenced teleost fish genomes retain 9-11 of IGFBP genes. This scenario is supported by the phylogenies of three adjacent gene families in the HOX gene regions, namely the epidermal growth factor receptors (EGFR) and the Ikaros and distal-less (DLX) transcription factors. Our sequence comparisons show that several important structural components in the IGFBPs are ancestral vertebrate features that have been maintained in all orthologs, for instance the integrin interaction motif Arg-Gly-Asp in IGFBP-2. In contrast, the Arg-Gly-Asp motif in IGFBP-1 has arisen independently in mammals. The large degree of retention of IGFBP genes after the ancient expansion of the gene family strongly suggests that each gene evolved distinct and important functions early in vertebrate evolution.

textabstractIn order to investigate potential changes in insulin-like growth factor binding proteins (IGFBPs) during human follicle maturation, we examined the IGFBP profiles in follicular fluid from follicles in different stages of maturation. Samples were obtained from ovaries of women with

AIM: To investigate whether kr(U)ppel-like factor 6 (KLF6)plays an important role in the development and/or progression of colorectal cancer.METHODS: A total of 123 formalin-fixed and paraffinembedded colorectal cancer specimens were analyzed by immunohistochemistry using tissue microarray for the expression of KLF6 protein. The specimens were collected over a 3-year period in the laboratories at our large teaching hospital in Seoul, Republic of Korea. The correlation of KLF6 expression with clinicopathologic parameters was analyzed by x2 test and Bartholomew test.RESULTS: Normal colonic epithelium showed weak to moderate expression of KLF6, whereas reduced KLF 6expression or loss of KLF6 expression was seen in 45(36.6%) of the 123 colorectal carcinoma specimens.Interestingly, aberrant expression of KLF6 was detected in 25 (43.1%) of 58 cases with metastasis to regional lymph node and in 31 (47.0%) of 66 tumors more than 5 cm in size. Statistically, loss of KLF6 expression was significantly associated with tumor size (P＜0.05).However, there was no significant correlation between KLF6 expression and Dukes' stage (Bartholomew test,P＞ 0.05), tumor location and lymph node metastasis (x2test, P＞ 0.05).CONCLUSION: Loss of KLF6 expression may be a common and early event in colorectal carcinogenesis.

AIM: To evaluate the effect of resistance to activated protein C (aPCR), the most common known inherited thrombophilic disorder, on the risk of intestinal operation of fibrostenosis in patients with Crohn's disease (CD).METHODS: In a previous study, we assessed the prevalence of aPCR in CD. In a retrospective casecontrolled study, 8 of these CD patients with aPCR were now compared with 24 CD patients without aPCR,matched by gender, age at diagnosis and duration of disease in a 1:3 fashion. The primary end point was the occurrence of an intestinal CD-related operation with evidence of fibrostenosis in the bowel resection specimen.RESULTS: The Kaplan-Meier analysis revealed that patients with aPCR had a lower probability of remaining free of operation with fibrostenosis than patients without aPCR (P = 0.0372; exact log-rank test) resulting in a significantly shorter median time interval from diagnosis of CD to the first operation with fibrostenosis (32 vs 160mo). At 10 years, the likelihood of remaining free of operation with fibrostenosis was 25% for patients with aPCR and 57.8% for patients without aPCR.CONCLUSION: CD patients with aPCR are at higher risk to undergo intestinal operation of fibrostenosis than those without aPCR. This supports our hypothesis of aPCR being a possible risk factor for fibrostenosis in CD.

The mechanisms driving bone marrow stem cell mobilization are poorly understood. A recent murine study found that circulating bone marrow-derived osteoprogenitor cells (MOPCs) were recruited to the site of recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone formation. Stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4 have been shown to mediate the homing of stem cells to injured tissues. We hypothesized that chemokines, such as SDF-1, are also involved with mobilization of bone marrow cells. The CD45(-) fraction is a major source of MOPCs. In this report we determined that the addition of BMP-2 or SDF-1 to collagen implants increased the number of MOPCs in the peripheral blood. BMP-2-induced mobilization was blocked by CXCR4 antibody, confirming the role of SDF-1 in mobilization. We determined for the first time that addition of SDF-1 to implants containing BMP-2 enhances mobilization, homing of MOPCs to the implant, and ectopic bone formation induced by suboptimal BMP-2 doses. These results suggest that SDF-1 increases the number of osteoprogenitor cells that are mobilized from the bone marrow and then home to the implant. Thus, addition of SDF-1 to BMP-2 may improve the efficiency of BMPs in vivo, making their routine use for orthopaedic applications more affordable and available to more patients.

Alcoholic liver injury comprises of interactions of various intracellular signaling events in the liver. Innate immune responses in the resident Kupffer cells of the liver, oxidative stress-induced activation of hepatocytes,fibrotic events in liver stellate cells and activation of liver sinusoidal endothelial cells all contribute to alcoholic liver injury. The signaling mechanisms associated with alcoholic liver injury vary based on the cell type involved and the extent of alcohol consumption. In this review we will elucidate the oxidative stress and signaling pathways affected by alcohol in hepatocytes and Kupffer cells in the liver by alcohol. The toll-like receptors and their down-stream signaling events that play an important role in alcohol-induced inflammation will be discussed. Alcohol-induced alterations of various intracellular transcription factors such as NFκB, PPARs and AP-1, as well as MAPK kinases in hepatocytes and macrophages leading to induction of target genes that contribute to liver injury will be reviewed. Finally, we will discuss the significance of heat shock proteins as chaperones and their functional regulation in the liver that could provide new mechanistic insights into the contributions of stress-induced signaling mechanisms in alcoholic liver injury.

Aim of this work was to investigate the ability of the antibodies against Surfactant proteins (SP) and Thyroid transcription factor 1 (TTF-1) to distinguish primary neoplasms of the lung from metastatic carcinomas to the lung and pleural mesotheliomas. We evaluated the immunohistochemical expression of the antibodies anti SP-A, SP-B, pro SP-C, SP-D, and TTF-1 in a series of 56 primary lung carcinomas, 9 metastatic carcinomas to the lung, 5 pleural mesotheliomas and 8 non-pulmonary carcinomas. Among primary lung neoplasms, only adenocarcinomas immunostained for all SP (specificity = 1; total sensitivity = 0.52). TTF-1 had an excellent specificity (= 1), but a weak sensitivity (= 0.34) in recognizing primary lung carcinomas. TTF-1 was present in lung adenocarcinomas which were negative for SPs; however it failed to distinguish the subtypes. Pleural mesotheliomas, pulmonary metastases and non-pulmonary carcinomas were not immunoreactive for SP-A, SP-B, SP-D, and TTF-1. Pro SP-C was positive also in the adenocarcinomas of the large bowel and in their pulmonary and nodal metastases. These results demonstrate that the combined use of antibodies anti SP-A, SP-B and TTF-1 is the best association in distinguishing primary lung carcinomas from metastatic carcinomas to the lung and pleural mesotheliomas.

The root cap covers the tip of the root and functions to protect the root from environmental stress. Cells in the last layer of the root cap are known as border cells, or border-like cells (BLCs) in Arabidopsis (Arabidopsis thaliana). These cells separate from the rest of the root cap and are released from its edge as a layer of living cells. BLC release is developmentally regulated, but the mechanism is largely unknown. Here, we show that the transcription factor NIN-LIKE PROTEIN7 (NLP7) is required for the proper release of BLCs in Arabidopsis. Mutations in NLP7 lead to BLCs that are released as single cells instead of an entire layer. NLP7 is highly expressed in BLCs and is activated by exposure to low pH, a condition that causes BLCs to be released as single cells. Mutations in NLP7 lead to decreased levels of cellulose and pectin. Cell wall-loosening enzymes such as CELLULASE5 (CEL5) and a pectin lyase-like gene, as well as the root cap regulators SOMBRERO and BEARSKIN1/2, are activated in nlp7-1 seedlings. Double mutant analysis revealed that the nlp7-1 phenotype depends on the expression level of CEL5 Mutations in NLP7 lead to an increase in susceptibility to a root-infecting fungal pathogen. Together, these data suggest that NLP7 controls the release of BLCs by acting through the cell wall-loosening enzyme CEL5.

Full Text Available Abstract Background Insulin-like growth factor binding protein 2 (IGFBP2 is overexpressed in ovarian malignant tissues and in the serum and cystic fluid of ovarian cancer patients, suggesting an important role of IGFBP2 in the biology of ovarian cancer. The purpose of this study was to assess the role of increased IGFBP2 in ovarian cancer cells. Results Using western blotting and tissue microarray analyses, we showed that IGFBP2 was frequently overexpressed in ovarian carcinomas compared with normal ovarian tissues. Furthermore, IGFBP2 was significantly overexpressed in invasive serous ovarian carcinomas compared with borderline serous ovarian tumors. To test whether increased IGFBP2 contributes to the highly invasive nature of ovarian cancer cells, we generated IGFBP2-overexpressing cells from an SKOV3 ovarian cancer cell line, which has a very low level of endogenous IGFBP2. A Matrigel invasion assay showed that these IGFBP2-overexpressing cells were more invasive than the control cells. We then designed small interference RNA (siRNA molecules that attenuated IGFBP2 expression in PA-1 ovarian cancer cells, which have a high level of endogenous IGFBP2. The Matrigel invasion assay showed that the attenuation of IGFBP2 expression indeed decreased the invasiveness of PA-1 cells. Conclusions We therefore showed that IGFBP2 enhances the invasion capacity of ovarian cancer cells. Blockage of IGFBP2 may thus constitute a viable strategy for targeted cancer therapy.

The lymphocyte transformation test (LTT) was given to 20 healthy subjects and 43 patients with generalized psoriasis vulgaris: it was given right after stimulation with PHA (spontaneous) and after stimulation with allogenic and autogenic proteinfactor (NPF). NPF was isolated from secondary lysosome granules of peripheral blood neutrophils. The results were analyzed using computer statistic tests. No distinct differences were noticed between the spontaneous transformation test in psoriatic patients compared to the controls. After stimulation with PHA, the percentage of blast cells was significantly lower in patients with psoriasis. When allogenic and autogenic NPF was used for stimulation, the LTT values were significantly higher in the psoriasis group than in the control subjects. This fact points out the increase in sensitivity of lymphocytes to NPF in active psoriasis and the possibility of abnormal neutrophil-lymphocyte interactions in vivo. This phenomenon may be intensified when under the influence of bacterial or viral agents, or medicaments; the degranulation of secondary lysosome granules of neutrophils occurs, causing the release of NPF. These investigations support our opinion that psoriasis is a systemic disease and that NPF plays a considerable role in the psoriatic reaction.

Full Text Available Kidney biopsy remains the mainstay of Lupus Nephritis (LN diagnosis and prognostication. The objective of this study is to identify non-invasive biomarkers that closely parallel renal pathology in LN. Previous reports have demonstrated that serum Insulin-like growth factor binding protein 4 (IGFBP-4 was increased in diabetic nephropathy in both animal models and patients. We proceeded to assess if IGFBP4 could be associated with LN. We performed ELISA using the serum of 86 patients with LN. Normal healthy adults (N = 23 and patients with other glomerular diseases (N = 20 served as controls. Compared to the healthy controls or other glomerular disease controls, serum IGFBP-4 levels were significantly higher in the patients with LN. Serum IGFBP-4 did not correlate well with systemic lupus erythematosus disease activity index (SLEDAI, renal SLEDAI or proteinuria, but it did correlate with estimated glomerular filtration rate (R = 0.609, P < 0.0001. Interestingly, in 18 patients with proliferative LN whose blood samples were obtained at the time of renal biopsy, serum IGFBP-4 levels correlated strongly with the chronicity index of renal pathology (R = 0.713, P < 0.001. IGFBP-4 emerges a potential marker of lupus nephritis, reflective of renal pathology chronicity changes.

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

Furfural from lignocellulosic hydrolysates is the key inhibitor for bio-ethanol fermentation. In this study, we report a strategy of improving the furfural tolerance in Zymomonas mobilis on the transcriptional level by engineering its global transcription sigma factor (σ(70), RpoD) protein. Three furfural tolerance RpoD mutants (ZM4-MF1, ZM4-MF2, and ZM4-MF3) were identified from error-prone PCR libraries. The best furfural-tolerance strain ZM4-MF2 reached to the maximal cell density (OD600) about 2.0 after approximately 30 h, while control strain ZM4-rpoD reached its highest cell density of about 1.3 under the same conditions. ZM4-MF2 also consumed glucose faster and yield higher ethanol; expression levels and key Entner-Doudoroff (ED) pathway enzymatic activities were also compared to control strain under furfural stress condition. Our results suggest that global transcription machinery engineering could potentially be used to improve stress tolerance and ethanol production in Z. mobilis.

Trefoil factor family (TFF) is composed of three secretory proteins (TFF1, TFF2 and TFF3) that play an important role in mucosal protection of gastrointestinal tract. Their overexpression in colorectal tumors seems to be associated with more aggressive disease. We collected serum samples from 79 healthy controls and 97 patients with metastatic colorectal cancer at the time of diagnosis or at progression. Serum levels of TTF1-3, CEA and CA19-9 were measured by ELISA. Serum TFF1 and TFF3 levels were significantly higher in patients with colorectal cancer compared to healthy controls (p TFF3 correlated with extent of liver involvement in patient without pulmonary metastases and patients with higher TFF3 levels had significantly worse outcome (p TFF3 had higher sensitivity and the same specificity. Our results indicate that TFF3 is an effective biomarker in patients with metastatic colorectal cancer with higher sensitivity than CEA a CA19-9. TFF3 levels strongly correlate with extension of liver disease and seem to have prognostic value.

Coleusin factor is a diterpenoid compound isolated from the root of a tropical plant, Coleus forskohlii. Although Coleusin factor has been reported to suppress proliferation of and induce apoptosis in several types of cancer cells, the effects of Coleusin factor on osteosarcoma and the underlying mechanism are still not fully understood. In this study, we show that Coleusin factor treatment potently inhibits the growth of osteosarcoma cells associated with G(1) cell-cycle arrest. Interestingly, apoptosis and cell death are not induced. Instead, Coleusin factor causes osteosarcoma cells to exhibit typical properties of differentiated osteoblasts, including a morphologic alteration resembling osteoblasts, the expression of osteoblast differentiation markers, elevated alkaline phosphatase activity, and increased cellular mineralization. Coleusin factor treatment significantly increases the expression of bone morphogenetic protein-2 (BMP-2), a crucial osteogenic regulator, and runt-related transcription factor 2 (RUNX2), one of the key transcription factors of the BMP pathway. When BMP-2 signaling is blocked, Coleusin factor fails to inhibit cell proliferation and to induce osteoblast differentiation. Thus, upregulation of BMP-2 autocrine is critical for Coleusin factor to induce osteoblast differentiation and exert its anticancer effects on osteosarcoma. Importantly, administration of Coleusin factor inhibits the growth of osteosarcoma xenografted in nude mice without systemic or immunologic toxicity. Osteosarcoma is a highly aggressive cancer marked by the loss of normal differentiation. Coleusin factor represents a new type of BMP-2 inducer that restores differentiation in osteosarcoma cells. It may provide a promising therapeutic strategy against osteosarcoma with minimal side effects.

) alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [(35)S]methionine, followed by protein separation on two-dimensional gel electrophoresis, displayed approximately 2500 proteins in the pI interval of 3-10. Treatment of TGF-beta(1) led to specific spot...... expression of alpha-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype....

AIM: To determine the effect of procedural and clinical factors upon C reactive protein(CRP) dynamics following transcatheter aortic valve implantation(TAVI).METHODS: Two hundred and eight consecutive patients that underwent transfemoral TAVI at two hospitals(Imperial, College Healthcare NHS Trust, Hammersmith Hospital, London, United Kingdom and San Raffaele Scientific Institute, Milan, Italy) were included. Daily venous plasma CRP levels were measured for up to 7 d following the procedure(or up to discharge). Procedural factors and 30-d safety outcomes according tothe Valve Academic Research Consortium 2 definition were collected. RESULTS: Following TAVI, CRP significantly increased reaching a peak on day 3 of 87.6 ± 5.5 mg/d L, P < 0.001. Patients who developed clinical signs and symptoms of sepsis had significantly increased levels of CRP(P < 0.001). The presence of diabetes mellitus was associated with a significantly higher peak CRP level at day 3(78.4 ± 3.2 vs 92.2 ± 4.4, P < 0.001). There was no difference in peak CRP release following balloonexpandable or self-expandable TAVI implantation(94.8 ± 9.1 vs 81.9 ± 6.9, P = 0.34) or if post-dilatation was required(86.9 ± 6.3 vs 96.6 ± 5.3, P = 0.42), however, when pre-TAVI balloon aortic valvuloplasty was performed this resulted in a significant increase in the peak CRP(110.1 ± 8.9 vs 51.6 ± 3.7, P < 0.001). The development of a major vascular complication did result in a significantly increased maximal CRP release(153.7 ± 11.9 vs 83.3 ± 7.4, P = 0.02) and there was a trend toward a higher peak CRP following major/lifethreatening bleeding(113.2 ± 9.3 vs 82.7 ± 7.5, P = 0.12) although this did not reach statistical significance. CRP was not found to be a predictor of 30-d mortality on univariate analysis. CONCLUSION: Careful attention should be paid to baseline clinical characteristics and procedural factors when interpreting CRP following TAVI to determine their future management.

Ricin (RCA60) is a potent cytotoxic protein with lectin domains, contained in the seeds of the castor bean Ricinus communis. It is a potential biohazard. To corroborate the biological properties of ricin, it is essential to understand the recognition factors involved in the ricin-glycotope interaction. In previous reports, knowledge of the binding properties of ricin was limited to oligosugars and glycopeptides with different specificities. Here, recognition factors of the lectin domains in ricin were examined by enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using mammalian Gal/GalNAc structural units and corresponding polyvalent forms. Except for blood group GalNAcalpha1-3Gal (A) active and Forssman (GalNAcalpha1-3GalNAc, F) disaccharides, ricin has a broad range of affinity for mammalian disaccharide structural units-Galbeta1-4Glcbeta1-(Lbeta), Galbeta1-4GlcNAc (II), Galbeta1-3GlcNAc (I), Galbeta1-3GalNAcalpha1-(Talpha), Galbeta1-3GalNAcbeta1-(Tbeta), Galalpha1-3Gal (B), Galalpha1-4Gal (E), GalNAcbeta1-3Gal (P), GalNAcalpha1-Ser/Thr (Tn) and GalNAcbeta1-4Gal (S). Among the polyvalent glycotopes tested, ricin reacted best with type II-containing glycoproteins (gps). It also reacted well with several T (Thomsen-Friedenreich), tumor-associated Tn and blood group Sd. (a+)-containing gps. Except for bird nest and Tamm-Horsfall gps (THGP), this lectin reacted weakly or not at all with ABH-blood type and sialylated gps. From the present and previous results, it can be concluded that: (i) the combining sites of these lectin domains should be a shallow-groove type, recognizing Galbeta1-4Glcbeta1- and Galbeta1-3(4)GlcNAcbeta- as the major binding site; (ii) its size may be as large as a tetrasaccharide and most complementary to lacto-N-tetraose (Galbeta1-3GlcNAc beta1-3Galbeta1-4Glc) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc); (iii) the polyvalency of glycotopes, in general, enhances binding; (iv) a hydrophobic interaction in the vicinity

A paleo-experimental evolution report on elongation factor EF-Tu structural stability results has provided an opportunity to rewind the tape of life using the ancestral protein sequence reconstruction modeling approach; consistent with the book of life dogma in current biology and being an important component in the astrobiology community. Fractal dimension via the Higuchi fractal method and Shannon entropy of the DNA sequence classification could be used in a diagram that serves as a simple summary. Results from biomedical gene research provide examples on the diagram methodology. Comparisons between biomedical genes such as EEF2 (elongation factor 2 human, mouse, etc), WDR85 in epigenetics, HAR1 in human specificity, DLG1 in cognitive skill, and HLA-C in mosquito bite immunology with EF Tu DNA sequences have accounted for the reported circular dichroism thermo-stability data systematically; the results also infer a relatively less volatility geologic time period from 2 to 3 Gyr from adaptation viewpoint. Comparison to Thermotoga maritima MSB8 and Psychrobacter shows that Thermus thermophilus HB8 EF-Tu calibration sequence could be an outlier, consistent with free energy calculation by NUPACK. Diagram methodology allows computer simulation studies and HAR1 shows about 0.5% probability from chimp to human in terms of diagram location, and SNP simulation results such as amoebic meningoencephalitis NAF1 suggest correlation. Extensions to the studies of the translation and transcription elongation factor sequences in Megavirus Chiliensis, Megavirus Lba and Pandoravirus show that the studied Pandoravirus sequence could be an outlier with the highest fractal dimension and lowest entropy, as compared to chicken as a deviant in the DNMT3A DNA methylation gene sequences from zebrafish to human and to the less than one percent probability in computer simulation using the HAR1 0.5% probability as reference. The diagram methodology would be useful in ancestral gene

Full Text Available The previous survey identified 70 basic helix-loop-helix (bHLH proteins, but it was proved to be incomplete, and the functional information and regulatory networks of frog bHLH transcription factors were not fully known. Therefore, we conducted an updated genome-wide survey in the Xenopus tropicalis genome project databases and identified 105 bHLH sequences. Among the retrieved 105 sequences, phylogenetic analyses revealed that 103 bHLH proteins belonged to 43 families or subfamilies with 46, 26, 11, 3, 15, and 4 members in the corresponding supergroups. Next, gene ontology (GO enrichment analyses showed 65 significant GO annotations of biological processes and molecular functions and KEGG pathways counted in frequency. To explore the functional pathways, regulatory gene networks, and/or related gene groups coding for Xenopus tropicalis bHLH proteins, the identified bHLH genes were put into the databases KOBAS and STRING to get the signaling information of pathways and protein interaction networks according to available public databases and known protein interactions. From the genome annotation and pathway analysis using KOBAS, we identified 16 pathways in the Xenopus tropicalis genome. From the STRING interaction analysis, 68 hub proteins were identified, and many hub proteins created a tight network or a functional module within the protein families.

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [35S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of Gαq but have no effect on stimulation of Gαi or Gαs. In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both Gαq and Gαi but decrease stimulation of Gαs. Consistent with these functional data, NHERF2 formed cellular complexes with both Gαq and Gαi, whereas NHERF1 was found to interact only with Gαq. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation. PMID:20562104

2,3-Dihydro-5-methyl-3-([morpholinyl]methyl)pyrollo(1,2,3-de)-1,4-benzoxazinyl]-[1-naphthaleny]methanone [WIN 55,212-2 (WIN)] is a synthetic cannabinoid that inhibits RKO, HT-29 and SW480 cell growth, induced apoptosis, and downregulated expression of survivin, cyclin D1, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and its receptor (VEGFR1). WIN also decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, and this is consistent with the observed downregulation of the aforementioned Sp-regulated genes. In addition, we also observed by RNA interference (RNAi) that the oncogenic cap protein eIF4E was an Sp-regulated gene also downregulated by WIN in colon cancer cells. WIN-mediated repression of Sp proteins was not affected by CB receptor antagonists or by knockdown of the receptor but was attenuated by the phosphatase inhibitor sodium orthovanadate or by knockdown of protein phosphatase 2A (PP2A). WIN-mediated repression of Sp1, Sp3 and Sp4 was due to PP2A-dependent downregulation of microRNA-27a (miR-27a) and induction of miR-27a-regulated ZBTB10 which has previously been characterized as an “Sp repressor”. The results demonstrate that the anticancer activity of WIN is due, in part, to PP2A-dependent disruption of miR-27a:ZBTB10 and ZBTB10-mediated repression of Sp transcription factors and Sp-regulated genes including eIF4E. PMID:24030632

The amino acid hypusine is formed by post-translational modification of a lysine residue in eukaryotes and archaebacteria but up to now only the eukaryotic translation initiation factor eIF-5A has been known to contain this unique component. We isolated and purified a hypusine-containing protein from the thermophilic archaebacterium Sulfolobus acidocaldarius. The mainly cytosolic protein comprised about 0.03% of the post-ribosomal supernatant protein. No other hypusine-containing protein could be detected in S. acidocaldarius. The molar ratio of hypusine/hypusine-containing protein was 1:1. SDS/PAGE showed a molecular mass of 16.8 kDa; a pI of 7.8 for the native protein resulted from IEF. The N-terminus was blocked. Four cyanogen bromide fragments were partially sequenced and used to derive two 17-base oligonucleotide probes. A 3-kb HindIII fragment of genomic DNA hybridizing with both probes was cloned. By sequencing of exonuclease III deletion clones an open reading frame of 405 nucleotides was found coding for a protein of 135 amino acids with a molecular mass of 15 kDa. It contained all cyanogen bromide sequences analysed. Sequence alignment revealed that seven of eight residues around Lys40 in the Sulfolobus hypusine-containing protein were identical to the nonapeptides centered by hypusine in the three eIF-5A proteins sequenced so far. The Edman procedure gave no phenylthiohydantoin derivative for this position. For a central region of 44 residues a sequence similarity of 54% between the archaebacterial and eukaryotic proteins was calculated; for the total sequence about 33% similarity resulted. In addition, there were a number of conservative changes. The unique lysine modification surrounded by a conserved sequence strongly suggests a common ancestry of archaebacterial hypusine-containing protein and eIF-5A. Together with similarities in molecular mass and intracellular localization, it may point to an analogous biochemical function.

Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized beta-galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years.

Full Text Available Abstract Background In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine resuscitation-promoting factor (Rpf is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria. The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria and obtain information about how they may control bacterial growth and resuscitation. Results In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we have called Sps. Although totally unrelated in both sequence and secondary structure, the Rpf and Sps domains fulfil the same function. We propose that these proteins have undergone "non-orthologous domain displacement", a phenomenon akin to "non-orthologous gene displacement" that has been described previously. Proteins containing the Sps domain are widely distributed throughout the firmicutes and they too fall into a number of distinct subfamilies. Comparative analysis of the accessory domains in the Rpf and Sps proteins, together with their weak similarity to lytic transglycosylases, provide clear evidence that they are muralytic enzymes. Conclusions The results indicate that the firmicute Sps proteins and the actinobacterial Rpf proteins are cognate and that they control bacterial culturability via enzymatic modification of the bacterial cell envelope.

ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we

Full Text Available Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory proteinfactor H (fH to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.

Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

Human sterile alpha motif domain-containing 9 (SAMD9) protein is a host restriction factor for poxviruses, but it can be overcome by some poxvirus host-range proteins that share homology with vaccinia virus C7 protein. To understand the mechanism of action for this important family of host-range factors, we determined the crystal structures of C7 and myxoma virus M64, a C7 family member that is unable to antagonize SAMD9. Despite their different functions and only 23% sequence identity, the two proteins have very similar overall structures, displaying a previously unidentified fold comprised of a compact 12-stranded antiparallel β-sandwich wrapped in two short α helices. Extensive structure-guided mutagenesis of C7 identified three loops clustered on one edge of the β sandwich as critical for viral replication and binding with SAMD9. The loops are characterized with functionally important negatively charged, positively charged, and hydrophobic residues, respectively, together forming a unique "three-fingered molecular claw." The key residues of the claw are not conserved in two C7 family members that do not antagonize SAMD9 but are conserved in distantly related C7 family members from four poxvirus genera that infect diverse mammalian species. Indeed, we found that all in the latter group of proteins bind SAMD9. Taken together, our data indicate that diverse mammalian poxviruses use a conserved molecular claw in a C7-like protein to target SAMD9 and overcome host restriction.

The vitellogenin-binding protein (VBP) is a member of the proline and acidic-region rich (PAR) family of bZip transcription factors. PAR is located N-terminally to the DNA-binding domain. VBP binds to specific sites within the 300-bp 5'-flanking region of the chicken-liver-specific estrogen-dependen

BACKGROUND: We aimed to examine the prevalence of and modifiable factors associated with elevated C-reactive Protein (CRP), a marker of inflammation, in men and women with newly diagnosed Type 2 Diabetes mellitus (DM) in a population-based setting. METHODS: CRP was measured in 1,037 patients (57%...

The objective was to derive factors to predict daily fat (F) and protein (P) yield or somatic cell score (SCS) when milk is sampled once for cows milked twice per d. Milk samples were collected for each milking on test-day by Dairy Herd Improvement personnel from herds recording milking times and m...

is composed of 9 to 11 nonidentical polypeptides; only 2 of these, with molecular weights of 120,000 and 70,000, were phosphorylated. A lower level of phosphorylation of initiation factor IF-E3 was found with the cyclic AMP-dependent protein kinase; the polypeptide of molecular weight 140,000 was the major...

foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress....

Enhancing factor (EF) protein, an isoform of secretory phospholipase A2 (PLA2), was purified as a modulator of epidermal growth factor from the small intestine of the Balb/c mouse. It was for the first time that a growth modulatory property of sPLA2 was demonstrated. Deletion mutation analysis of EF cDNA carried out in our laboratory showed that enhancing activity and phospholipase activity are two separate activities that reside in the same molecule. In order to study the specific amino acids involved in each of these activities, two site-directed mutants of EF were made and expressed in vitro. Comparison of enhancing activity as well as phospholipase A2 activity of these mutant proteins with that of wild type protein helped in identification of some of the residues important for both the activities.

The function of S100A4, a member of the calcium-binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro-metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms...... and protein partners (receptors) has remained elusive. To identify putative targets for extracellular S100A4, we screened a phage display peptide library using S100A4 as bait. We identified three independent peptide motifs with varying affinities for the S100A4 protein. Sequence analyses indicated...... that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4...

Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair. Objective: To determine the role of the AMPKα2 subunit in vascular repair. Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2−/− versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice. Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia. PMID:27777247

Full Text Available Objective(s Staphylococcus aureus is a leading cause of many nosocomial and community acquired infections. According to many reports, antibiotic therapy can not guarantee the eradication of S. aureus infections. Thus designing an adhesin based vaccine could restrain the S. aureus infections. This study designed for construction of a new fusion protein vaccine against S. aureus infections based on adhesin molecules fibronectin binding protein A (FnBPA and clumping factor A (ClfA. Materials and Methods Bioinformatic experiments were performed using Oligo analyzer and DNAMAN softwares. The fragments corresponding to fnbA binding domain and a C-terminal fragment from clfA were amplified from S. aureus NCTC8325 genomic DNA. Purified PCR products and the vector, pET15b, were digested with NcoI and BamHI. The digested PCR products were hybridized together and then ligated to digested vector. Finally incomplete construct was assembled by Taq DNA polymerase. To quick confirmation of cloning procedure the new construct designated pfnbA-clfA was digested with NcoI and BamHI. To further verification, the product was sent for sequencing. Results The data based on bioinformatic analysis showed no homology between fusion protein and human proteins. Digestion of new vector with NcoI and BamHI confirmed the ligation of fusion protein sequence into pET15b. Sequencing results verified the integrity of target sequences. Conclusion This study is the first effort to construct a new fusion protein vector based on S. aureus adhesins using a new design. This project is being continued to study the expression and biological activity of the fusion protein in a cell culture model.

Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proliferation of normal cells under pathophysiological conditions. In order to evaluate its potential as a pharmaceutical target, the protein must be produced and purified in sufficiently high yields. In the present study, we report the high level expression of Tpr protein of R. norvegicus employing heterologous host, Escherichia coli, to permit its structural characterization in great detail. We here demonstrate that the Tpr protein was expressed in soluble form and approximately 90 mg/L of the purified protein at the shake flask level could be achieved to near homogeneity using single step-metal chelate affinity chromatography. The amino acid sequence of the protein was confirmed by mass spectroscopic analysis. The highly unstable and disordered Tpr protein was imparted structural and functional stability by the addition of glycerol and it has been shown that the natively unfolded Tpr protein retains DNA binding ability under these conditions only. Thus, the present study emphasizes the significance of an efficient prokaryotic system, which results in a high level soluble expression of a DNA binding protein of eukaryotic origin. Thus, the present strategy employed for purification of the R. norvegicus Tpr protein bypasses the need for the tedious expression strategies associated with the eukaryotic expression systems.

There is evidence that the expression of members of the fibroblast growth factor (FGF) protein family is altered in post-mortem brains of humans suffering from major depressive disorder. The present study examined whether the expression of fibroblast growth factor-2 (FGF2) and fibroblast growth factor receptor-1 (FGFR1) protein is altered following chronic stress in an animal model. Rats were exposed to 35 days of chronic unpredictable mild stress, and then tested using open-field and sucrose consumption tests. Compared with the control group, rats in the chronic stress group exhibited obvious depressive-like behaviors, including anhedonia, anxiety and decreased mobility. The results of western blot analysis and immunohistochemical analysis revealed a downregulation of the expression of FGF2 and FGFR1 in the hippocampus of rats, particularly in the CA1, CA3 and dentate gyrus. This decreased expression is in accord with the results of post-mortem studies in humans with major depressive disorder. These findings suggest that FGF2 and FGFR1 proteins participate in the pathophysiology of depressive-like behavior, and may play an important role in the mechanism of chronic stress-induced depression.

Maternal diet during pregnancy is one of the most important factors associated with adequate fetal growth. There are many complications associated with fetal growth restriction that lead to lifelong effects. The aim of this review was to describe the studies examining the effects of protein energy supplementation during pregnancy on fetal growth focusing on the contextual differences. Relevant articles published between 2007 and 2012 were identified through systematic electronic searches of the PubMed, Science Direct, and EBSCO database and the examination of the bibliographies of retrieved articles. The search aimed to identify studies examining pregnant women receiving protein and/or energy during pregnancy and to assess fetal growth measures. Data of effectiveness and practical aspects of protein energy supplementation during pregnancy were extracted and compiled. Twenty studies (11 randomized controlled trials, 8 controlled before and after, and 1 prospective study) were included in this review. Positive outcomes in infants and women cannot be expected if the supplementation is not needed. Therefore, it is essential to correctly select women who will benefit from dietary intervention programs during pregnancy. However, there is currently no consensus on the most effective method of identifying these women. The content of protein in the supplements considering total diet is also an important determinant of fetal growth. Balanced protein energy supplementation (containing up to 20% of energy as protein) given to pregnant women with energy or protein deficit appears to improve fetal growth, increase birth weight (by 95-324 g) and height (by 4.6-6.1 mm), and decrease the percentage of low birth weight (by 6%). Supplements with excess protein (>20% of energy as protein) provided to women with a diet already containing adequate protein may conversely impair fetal growth. There is also no consensus on the best time to start supplementation. Strong quality studies

Background Maternal diet during pregnancy is one of the most important factors associated with adequate fetal growth. There are many complications associated with fetal growth restriction that lead to lifelong effects. The aim of this review was to describe the studies examining the effects of protein energy supplementation during pregnancy on fetal growth focusing on the contextual differences. Methods Relevant articles published between 2007 and 2012 were identified through systematic electronic searches of the PubMed, Science Direct, and EBSCO database and the examination of the bibliographies of retrieved articles. The search aimed to identify studies examining pregnant women receiving protein and/or energy during pregnancy and to assess fetal growth measures. Data of effectiveness and practical aspects of protein energy supplementation during pregnancy were extracted and compiled. Results Twenty studies (11 randomized controlled trials, 8 controlled before and after, and 1 prospective study) were included in this review. Positive outcomes in infants and women cannot be expected if the supplementation is not needed. Therefore, it is essential to correctly select women who will benefit from dietary intervention programs during pregnancy. However, there is currently no consensus on the most effective method of identifying these women. The content of protein in the supplements considering total diet is also an important determinant of fetal growth. Balanced protein energy supplementation (containing up to 20% of energy as protein) given to pregnant women with energy or protein deficit appears to improve fetal growth, increase birth weight (by 95–324 g) and height (by 4.6–6.1 mm), and decrease the percentage of low birth weight (by 6%). Supplements with excess protein (>20% of energy as protein) provided to women with a diet already containing adequate protein may conversely impair fetal growth. There is also no consensus on the best time to start

Full Text Available Abstract Background Lipoprotein (a (LP (a is an independent cardiovascular risk factor that is not widely studied in people of sub-Saharan African origin. The aim of this report is to determine the frequency of occurrence of elevated Lp (a and possible relationship with total cholesterol (TCHOL, high density lipoprotein cholesterol (HDL-C, low density lipoprotein cholesterol (LDL-C, triglycerides (TG, C reactive protein (CRP and serum uric acid (SUA. Methods This is a cross sectional study carried out in 200 Nigerian patients with type 2 DM and 100 sex and age matched healthy Controls aged between 32-86 years. We determined the frequency of occurrence of elevated Lp (a levels in the study subjects and compared clinical and biochemical variables between type 2 diabetic patients and non-diabetic patients. Clinical and biochemical parameters were also compared between subjects with type 2 DM who had elevated LP (a and normal LP (a levels. Long term glycaemic control using glycosylated haemoglobin was determined and compared in the study subjects. Test statistics used include chi square, correlation coefficient analysis and Student's t test. Results The mean Lp(a concentration differed significantly between type 2 diabetic patients and the Control subjects (18.7 (5.8 mg/dl vs 23 (6.8 mg/dl, 0.00001. Similarly, the prevalence of high LP (a levels in type 2 DM patients was significantly higher than that of the Control subjects (12.5% vs 4%, p-0.019. The mean levels of the lipid profile parameters (TCHOL, LDL-C, TG, LDL/HDL and CRP were significantly higher in DM patients than in the Control subjects. The mean LP (a levels were comparable in both sexes and in DM subjects with and without hypertension. TG was the only parameter that differed significantly between subjects with elevated Lp (a levels and those with normal Lp (a levels. There was a significant positive correlation (r between Lp(a levels and TG, LDL-C. TCHOL, LDL/HDL and uric acid. No

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.

A series of experiments was performed to evaluate the influence of a number of physico-chemical factors on the diffusion of a model protein, bovine serum albumin (BSA), from dried chitosan-coated alginate microcapsules. Diffusion of BSA was quantified during the microcapsule manufacture processes (gelation, washing, rinsing) and during incubation in conditions simulating the pH encountered during the gastric (0.1 N HCl; pH 1.5) and intestinal (200 mM Tris-HCl; pH 7.5) phases of digestion. Factors tested included alginate and chitosan concentration, calcium chloride (CaCl2) concentration in the gelation medium, loading rate, chitosan molecular mass and pH of the gelation medium. Microcapsule size and gelation time were altered in order to determine their effects on protein retention. Alginate and chitosan concentration significantly influenced BSA retention during microcapsule manufacture and acid incubation, as did calcium chloride concentration in the gelation medium (P<0.05). BSA retention during manufacture was not significantly altered by protein loading rate or pH of the encapsulation medium, however, protein retention during acid incubation decreased significantly with increasing protein loading rate and encapsulation medium pH (P<0.05). Microcapsules that were washed with acetone following manufacture demonstrated significantly increased protein retention during acid incubation (P<0.05). In microcapsules that had been acetone-dried to a point whereby their mass was reduced to 10% of that immediately following encapsulation, protein retention was over 80% following 24-h acid incubation vs. only 20% protein retention from non acetone-dried microcapsules. The presence of calcium in the neutral buffer medium significantly reduced BSA diffusion in a concentration-dependent manner (P<0.05).

Full Text Available Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA. Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins and ribosome precursors (pre-ribosomes are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.

Full Text Available In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs, which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1 and C. albicans (Cph2 have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1 and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

Highlights: Black-Right-Pointing-Pointer Mxi1 regulates cell proliferation. Black-Right-Pointing-Pointer Expression of IGFBP-3 is regulated by Mxi1. Black-Right-Pointing-Pointer Inactivation of Mxi1 reduces IGFBP-3 expression in vitro and in vivo. -- Abstract: Mxi1, a member of the Myc-Max-Mad network, is an antagonist of the c-Myc oncogene and is associated with excessive cell proliferation. Abnormal cell proliferation and tumorigenesis are observed in organs of Mxi1-/- mice. However, the Mxi1-reltaed mechanism of proliferation is unclear. The present study utilized microarray analysis using Mxi1 mouse embryonic fibroblasts (MEFs) to identify genes associated with cell proliferation. Among these genes, insulin-like growth factor binding protein-3 (IGFBP-3) was selected as a candidate gene for real-time PCR to ascertain whether IGFBP-3 expression is regulated by Mxi1. Expression of IGFBP-3 was decreased in Mxi1-/- MEFs and Mxi1-/- mice, and the gene was regulated by Mxi1 in Mxi1 MEFs. Furthermore, proliferation pathways related to IGFBP-3 were regulated in Mxi1-/- mice compared to Mxi1+/+ mice. To determine the effect of Mxi1 inactivation on the induction of cell proliferation, a proliferation assay is performed in both Mxi1 MEFs and Mxi1 mice. Cell viability was regulated by Mxi1 in Mxi1 MEFs and number of PCNA-positive cells was increased in Mxi1-/- mice compared to Mxi1+/+ mice. Moreover, the IGFBP-3 level was decreased in proliferation defect regions in Mxi1-/- mice. The results support the suggestion that inactivation of Mxi1 has a positive effect on cell proliferation by down-regulating IGFBP-3.

The neurobiological mechanisms of emotional modulation and the molecular pathophysiology of anxiety disorders are largely unknown. The fibroblast growth factor (FGF) family has been implicated in the regulation of many physiological and pathological processes, which include the control of emotional behaviors. The present study examined mice with a targeted deletion of the fgf-bp3 gene, which encodes a novel FGF-binding protein, in animal models relevant to anxiety. To define the behavioral consequences of FGF-BP3 deficiency, we evaluated fgf-bp3-deficient mice using anxiety-related behavioral paradigms that provide a conflict between the desire to explore an unknown area or objects and the aversion to a brightly lit open space. The fgf-bp3-deficient mice exhibited alterations in time spent in the central area of the open-field arena, were less active in the lit areas of a light/dark transition test, and had a prolonged latency to feed during a novelty-induced hypophagia test. These changes were associated with alterations in light-induced orbitofrontal cortex (OFC) activation in an extracellular signal-regulated kinase (ERK) pathway-dependent manner. These results demonstrate that FGF-BP3 is a potent mediator of anxiety-related behaviors in mice and suggest that distinct pathways regulate emotional behaviors. Therefore, FGF-BP3 plays a critical role in the regulation of emotional states and in the development of anxiety disorders and should be investigated as a therapeutic target for anxiety disease in humans.

Brain-derived neurotrophic factor (BDNF) and its cognate receptor tyrosine kinase B (TrkB) play important roles in regulating survival, structure, and function of CNS neurons. One method of studying the functions of these molecules has utilized in vitro hippocampal slice preparations. An important caveat to using slices, however, is that slice preparation itself might alter the expression of BDNF, thereby confounding experimental results. To address this concern, BDNF immunoreactivity was examined in rodent slices using two different methods of slice preparation. Rapid and anatomically selective regulation of BDNF content followed slice preparation using both methodologies; however, different patterns of altered BDNF immunoreactivity were observed. First, in cultured slices, BDNF content decreased in the dentate molecular layer and increased in the CA3 pyramidal cell layer and the mossy fiber pathway of the hippocampus after 30 min. Furthermore, an initially "punctate" pattern of BDNF labeling observed in the mossy fiber pathway of control sections changed to homogenous labeling of the pathway in vitro. In contrast to these findings, slices prepared as for acute slice physiology exhibited no change in BDNF content in the molecular layer and mossy fiber pathway 30 min after slicing, but exhibited significant increases in the dentate granule and CA3 pyramidal cell layers. These findings demonstrate that BDNF protein content is altered following slice preparation, that different methods of slice preparation produce different patterns of BDNF regulation, and raise the possibility that BDNF release and TrkB activation may also be regulated. These consequences of hippocampal slice preparation may confound analyses of exogenous or endogenous BDNF on hippocampal neuronal structure or function.

Full Text Available The signaling outputs of Receptor Tyrosine Kinases, G-protein coupled receptors and integrins converge to mediate key cell process such as cell adhesion, cell migration, cell invasion and cell proliferation. Once activated by their ligands, these cell surface proteins recruit and direct a diverse range of proteins to disseminate the appropriate response downstream of the specific environmental cues. One of the key groups of proteins required to regulate these activities is the family of serine/threonine intracellular kinases called Protein Kinase Cs. The activity and subcellular location of PKCs are mediated by a series of tightly regulated events and is dependent on several posttranslational modifications and the availability of second messengers. Protein Kinase Cs exhibit both pro- and anti-tumorigenic effects making them an interesting target for anti-cancer treatment.

The signaling outputs of Receptor Tyrosine Kinases, G-protein coupled receptors and integrins converge to mediate key cell process such as cell adhesion, cell migration, cell invasion and cell proliferation. Once activated by their ligands, these cell surface proteins recruit and direct a diverse range of proteins to disseminate the appropriate response downstream of the specific environmental cues. One of the key groups of proteins required to regulate these activities is the family of serine/threonine intracellular kinases called Protein Kinase Cs. The activity and subcellular location of PKCs are mediated by a series of tightly regulated events and is dependent on several posttranslational modifications and the availability of second messengers. Protein Kinase Cs exhibit both pro- and anti-tumorigenic effects making them an interesting target for anti-cancer treatment.

Borrelia spielmanii sp. nov. has recently been shown to be a novel human pathogenic genospecies that causes Lyme disease in Europe. In order to elucidate the immune evasion mechanisms of B. spielmanii, we compared the abilities of isolates obtained from Lyme disease patients and tick isolate PC-Eq17 to escape from complement-mediated bacteriolysis. Using a growth inhibition assay, we show that four B. spielmanii isolates, including PC-Eq17, are serum resistant, whereas a single isolate, PMew, was more sensitive to complement-mediated lysis. All isolates activated complement in vitro, as demonstrated by covalent attachment of C3 fragments; however, deposition of the later activation products C6 and C5b-9 was restricted to the moderately serum-resistant isolate PMew and the serum-sensitive B. garinii isolate G1. Furthermore, serum adsorption experiments revealed that all B. spielmanii isolates acquired the host alternative pathway regulators factor H and factor H-like protein (FHL-1) from human serum. Both complement regulators retained their factor I-mediated C3b inactivation activities when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding proteins, BsCRASP-1 and BsCRASP-2, were identified, which we estimated to be approximately 23 to 25 kDa in mass. A further factor H binding protein, BsCRASP-3, was found exclusively in the tick isolate, PC-Eq17. This is the first report describing an immune evasion mechanism utilized by B. spielmanii sp. nov., and it demonstrates the capture of human immune regulators to resist complement-mediated killing.

in protein networks driving the development of specific anatomical structures (e.g., outflow tract, ventricular septum, and atrial septum) that are malformed by CHD. This integrative analysis of CHD risk factors and responses suggests a complex pattern of functional interactions between genomic variation...... CHD risk factors and responses, we compiled and integrated comprehensive datasets from studies of CHD in humans and model organisms. We examined two alternative models of potential functional relationships between genes in these datasets: direct convergence, in which CHD risk factors significantly...... and directly impact the same genes and molecules and functional convergence, in which risk factors significantly impact different molecules that participate in a discrete heart development network. We observed no evidence for direct convergence. In contrast, we show that CHD risk factors functionally converge...

Full Text Available Objective: to study the time course of changes and an association of contact factors and their inhibitors with the global values of hemocoagulation, fibrinolysis, and inflammatory reactants in acute severe brain injury (SBI in order to deepen notions of Hageman factor system functioning. Subjects and methods. One hundred and thirteen patients with SBI were examined on 1 to 21 days of injury. The level of unconsciousness averaged a Glasgow coma score of 6.8±0.25. A control group included 23 healthy individuals. The investigators determined the activity of contact factors (prekallikrein, high-molecular-weight kininogen, factors XII, XI and their inhibitors (total activity of the protein C system, the activity and quantity of antithrombin III, C1 esterase inhibitor, a^antitrypsin, fl^-antiplasmin, fl^-macroglobulin, hemostatic parameters (blood fibrinolytic activity by an euglobulin test; factor XII-kallikrein-dependent fibrinolysis, treptokinase induced fibrinolysis by calculating the plasminogen reserve index, activated partial thromboplastin time, fibrinogen, D-dimer, and soluble fibrin monomer complexes, and inflammatory reactants (C-reactive protein, IL10, IL2, IL4, IL5, IL6, IL8, IL10, IL12p70, TNF-a, and IFN-y. Results. The acute period of SBI was marked by significant deficiency and imbalance of contact factors and their physiological inhibitors. In SBI, prekallikrein rather than factor XII plays a central role in the function of the contact factor system due to inflammatory inhibition of Hageman factor synthesis, which disturbs its key role in the reactions of contact activation of homeostatic proteolytic systems. Out of the considered systems, the activation of which is associated with contact factors, the function of the internal mechanism of fibrinolysis is largely changed; at the same time the internal hemocoagulation activation pathway remains virtually intact. When an inflammatory reaction develops after SBI, normal Hageman factor

Full Text Available Human granulocyte colony-stimulating factor (hGCSF, a neutrophil-promoting cytokine, is an effective therapeutic agent for neutropenia patients who have undergone several cancer treatments. Efficient production of hGCSF using E. coli is challenging because the hormone tends to aggregate and forms inclusion bodies. This study examined the ability of seven different N-terminal fusion tags to increase expression of soluble hGCSF in E. coli. Four tag proteins, namely maltose-binding protein (MBP, N-utilization substance protein A, protein disulfide isomerase (PDI, and the b'a' domain of PDI (PDIb'a', increased the solubility of hGCSF under normal conditions. Lowering the expression temperature from 30°C to 18°C also increased the solubility of thioredoxin-tagged and glutathione S-transferase-tagged hGCSF. By contrast, hexahistidine-tagged hGCSF was insoluble at both temperatures. Simple conventional chromatographic methods were used to purify hGCSF from the overexpressed PDIb'a'-hGCSF and MBP-hGCSF proteins. In total, 11.3 mg or 10.2 mg of pure hGCSF were obtained from 500 mL cultures of E. coli expressing PDIb'a'-hGCSF or MBP-hGCSF, respectively. SDS-PAGE analysis and silver staining confirmed high purity of the isolated hGCSF proteins, and the endotoxin levels were less than 0.05 EU/µg of protein. Subsequently, the bioactivity of the purified hGCSF proteins similar to that of the commercially available hGCSF was confirmed using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50s of the cell proliferation dose-response curves for hGCSF proteins purified from MBP-hGCSF and PDIb'a'-hGCSF were 2.83±0.31 pM, and 3.38±0.41 pM, respectively. In summary, this study describes an efficient method for the soluble overexpression and purification of bioactive hGCSF in E. coli.

Human granulocyte colony-stimulating factor (hGCSF), a neutrophil-promoting cytokine, is an effective therapeutic agent for neutropenia patients who have undergone several cancer treatments. Efficient production of hGCSF using E. coli is challenging because the hormone tends to aggregate and forms inclusion bodies. This study examined the ability of seven different N-terminal fusion tags to increase expression of soluble hGCSF in E. coli. Four tag proteins, namely maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), increased the solubility of hGCSF under normal conditions. Lowering the expression temperature from 30°C to 18°C also increased the solubility of thioredoxin-tagged and glutathione S-transferase-tagged hGCSF. By contrast, hexahistidine-tagged hGCSF was insoluble at both temperatures. Simple conventional chromatographic methods were used to purify hGCSF from the overexpressed PDIb'a'-hGCSF and MBP-hGCSF proteins. In total, 11.3 mg or 10.2 mg of pure hGCSF were obtained from 500 mL cultures of E. coli expressing PDIb'a'-hGCSF or MBP-hGCSF, respectively. SDS-PAGE analysis and silver staining confirmed high purity of the isolated hGCSF proteins, and the endotoxin levels were less than 0.05 EU/µg of protein. Subsequently, the bioactivity of the purified hGCSF proteins similar to that of the commercially available hGCSF was confirmed using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50s of the cell proliferation dose-response curves for hGCSF proteins purified from MBP-hGCSF and PDIb'a'-hGCSF were 2.83±0.31 pM, and 3.38±0.41 pM, respectively. In summary, this study describes an efficient method for the soluble overexpression and purification of bioactive hGCSF in E. coli.

Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

Full Text Available EF-Tu proteins of plastids, mitochondria, and the cytosolic counterpart EF-1α in plants, as well as EF-Tu proteins of bacteria, are highly conserved and multifunctional. The functions of EF-Tu include transporting the aminoacyl-tRNA complex to the A site of the ribosome during protein biosynthesis; chaperone activity in protecting other proteins from aggregation caused by environmental stresses, facilitating renaturation of proteins when conditions return to normal; displaying a protein disulfide isomerase activity; participating in the degradation of N-terminally blocked proteins by the proteasome; eliciting innate immunity and triggering resistance to pathogenic bacteria in plants; participating in transcription when an E. coli host is infected with phages. EF-Tu genes are upregulated by abiotic stresses in plants, and EF-Tu plays important role in stress responses. Expression of a plant EF-Tu gene confers heat tolerance in E. coli, maize knock-out EF-Tu null mutants are heat susceptible, and over-expression of an EF-Tu gene improves heat tolerance in crop plants. This review paper summarizes the current knowledge of EF-Tu proteins in stress responses in plants and progress on application of EF-Tu for developing crop varieties tolerant to abiotic stresses, such as high temperatures.

Full Text Available Abstract Background We have incorporated Bayesian model regularization with biophysical modeling of protein-DNA interactions, and of genome-wide nucleosome positioning to study protein-DNA interactions, using a high-throughput dataset. The newly developed method (BayesPI includes the estimation of a transcription factor (TF binding energy matrices, the computation of binding affinity of a TF target site and the corresponding chemical potential. Results The method was successfully tested on synthetic ChIP-chip datasets, real yeast ChIP-chip experiments. Subsequently, it was used to estimate condition-specific and species-specific protein-DNA interaction for several yeast TFs. Conclusion The results revealed that the modification of the protein binding parameters and the variation of the individual nucleotide affinity in either recognition or flanking sequences occurred under different stresses and in different species. The findings suggest that such modifications may be adaptive and play roles in the formation of the environment-specific binding patterns of yeast TFs and in the divergence of TF binding sites across the related yeast species.

Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

There is limited information on the influence of genetic and environmental variability on soybean protein composition. This study aimed to determine the role of genotype (G), environments (E), and the interrelationship of genotype and environment (G×E) on soybean seed protein. Three sets of nine soybean genotypes were grown in replicated trials at Maryland, South Carolina, and South Dakota. At each location, the nine genotypes were grown with two planting/sowing dates. We applied two-dimensional gel electrophoresis and mass spectrometry to study the variability of soybean storage and allergen proteins. Statistical analysis of 47 storage and 8 allergen proteins, in terms of differentially expressed protein spots significant at the p<0.005 level, was performed. We found more spots that showed statistically significant differences in expression among E compared to G and G×E interaction.

This is the first report, to our knowledge, to reveal important factors by which members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), squash (C. pepo), and zucchini (C. pepo), are selectively polluted with highly toxic hydrophobic contaminants, including organochlorine insecticides and dioxins. Xylem sap of C. pepo ssp. pepo, which is a high accumulator of hydrophobic compounds, solubilized the hydrophobic compound pyrene into the aqueous phase via some protein(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of xylem sap of two C. pepo subspecies revealed that the amount of 17-kD proteins in C. pepo ssp. pepo was larger than that in C. pepo ssp. ovifera, a low accumulator, suggesting that these proteins may be related to the translocation of hydrophobic compounds. The protein bands at 17 kD contained major latex-like proteins (MLPs), and the corresponding genes MLP-PG1, MLP-GR1, and MLP-GR3 were cloned from the C. pepo cultivars Patty Green and Gold Rush. Expression of the MLP-GR3 gene in C. pepo cultivars was positively correlated with the band intensity of 17-kD proteins and bioconcentration factors toward dioxins and dioxin-like compounds. Recombinant MLP-GR3 bound polychlorinated biphenyls immobilized on magnetic beads, whereas recombinant MLP-PG1 and MLP-GR1 did not. These results indicate that the high expression of MLP-GR3 in C. pepo ssp. pepo plants and the existence of MLP-GR3 in their xylem sap are related to the efficient translocation of hydrophobic contaminants. These findings should be useful for decreasing the contamination of fruit of the Cucurbitaceae family as well as the phytoremediation of hydrophobic contaminants.

We investigated how myofibrillar protein synthesis (MPS) and muscle anabolic signalling were affected by resistance exercise at 20-90% of 1 repetition maximum (1 RM) in two groups (25 each) of post-absorptive, healthy, young (24 +/- 6 years) and old (70 +/- 5 years) men with identical body mass indices (24 +/- 2 kg m(-2)). We hypothesized that, in response to exercise, anabolic signalling molecule phosphorylation and MPS would be modified in a dose-dependant fashion, but to a lesser extent in older men. Vastus lateralis muscle was sampled before, immediately after, and 1, 2 and 4 h post-exercise. MPS was measured by incorporation of [1,2-(13)C] leucine (gas chromatography-combustion-mass spectrometry using plasma [1,2-(13)C]alpha-ketoisocaparoate as surrogate precursor); the phosphorylation of p70 ribosomal S6 kinase (p70s6K) and eukaryotic initiation factor4Ebindingprotein 1 (4EBP1) was measured using Western analysis with anti-phosphoantibodies. In each group, there was a sigmoidal dose-response relationship between MPS at 1-2 h post-exercise and exercise intensity, which was blunted (P men. At all intensities, MPS fell in both groups to near-basal values by 2-4 h post-exercise. The phosphorylation of p70s6K and 4EBP1 at 60-90% 1 RM was blunted in older men. At 1 h post-exercise at 60-90% 1 RM, p70s6K phosphorylation predicted the rate of MPS at 1-2 h post-exercise in the young but not in the old. The results suggest that in the post-absorptive state: (i) MPS is dose dependant on intensity rising to a plateau at 60-90% 1 RM; (ii) older men show anabolic resistance of signalling and MPS to resistance exercise.

Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

A possible role of bovine platelets in the surface-mediated activation of Factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine plasma. The washed platelets before and after aggregation by ADP, thrombin or collagen did not show any ability to trigger or accelerate the activation of Factor XII and prekallikrein. On the contrary, these aggregates showed a potent inhibitory activity on the activation of those zymogens triggered by kaolin, amylose sulfate and sulfatide. The inhibitory substances from the supernatant of the thrombin-induced aggregates were separated into two major fractions, a low affinity fraction and a high affinity fraction, on a heparin-Sepharose column. The high affinity protein was identified as platelet factor 4, based on the amino acid composition. From the low affinity fraction, a beta-thromboglobulin (beta-TG)-like substance and three kinds of unknown proteins, named LA1, LA2, and LA3, were isolated by gel-filtration on a column of Sephadex G-100 or Sephadex G-75 followed by chromatography on a column of Mono S. The molecular weights of LA1, LA2, and LA3 were estimated to be 35,000, 26,000, and 11,000, respectively, on SDS-PAGE. LA2 was identified as a carbohydrate-less LA1, as judged from the amino acid composition and carbohydrate content. The inhibitory activities of these five cationic proteins on the activation of Factor XII and prekallikrein mediated with amylose sulfate, sulfatide and kaolin were different from each other. In the case of kaolin-mediated activation, LA3 was the most potent inhibitor, while platelet factor 4 and beta-TG-like substance did not show any significant inhibitory activity. Moreover, the inhibitory activities of all the cationic proteins were not correlated with their anti-heparin activities. Since these proteins were rapidly liberated from platelets by the action of the stimulants, the present results demonstrate a negative role of platelets in

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

AIM: To investigate the selective cytotoxic effect of constructed hybrid protein on cells expressing granulocyte macrophage colony stimulating factor (GM-CSF) receptor.METHODS: HepG2 (human hepatoma) and LS174T (coIon carcinoma) were used in this study. The fused gene was induced with 0.02% of arabinose for 4 h and the expressed protein was detected by Western blotting. The chimeric protein expressed in E. coli was checked for its cytotoxic activity on these cells and apoptosis was measured by comet assay and nuclear staining. RESULTS: The chimeric protein was found to be cytotoxic to the colon cancer cell line expressing GM-CSFRs,but not to HepG2 lacking these receptors. Maximum activity was observed at the concentration of 40 ng/mL after 24 h incubation. The IC50 was 20±3.5 ng/mL.CONCLUSION: Selective cytotoxic effect of the hybrid protein on the colon cancer cell line expressing GMCSF receptors (GM-CSFRs) receptor and apoptosis can be observed in this cell line. The hybrid protein can be considered as a therapeutic agent.

Highlights: ► Both R9Sox2 and Sox2 bind heparin with comparable affinity. ► Both R9Sox2 and Sox2 bind to fibroblasts, but only R9Sox2 is internalized. ► Internalization efficiency of R9Sox2 is 0.3% of the administered protein. ► Heparan sulfate adsorption may be part of a mechanism for managing cell death. -- Abstract: The binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD. However, only the R9Sox2 fusion protein is internalized by the cells. These results collectively show that binding to heparan sulfate is not sufficient for cellular uptake, thereby supporting a recent hypothesis that other proteins play a role in cell internalization of PTD-conjugated proteins.

Tumor necrosis factor (TNF)-α inhibitor has been shown to affect the periodontal condition of patients with rheumatoid arthritis (RA). The aim of the present study is to assess the effect of a fully humanized anti-TNF-α monoclonal antibody, adalimumab (ADA), on the periodontal condition of patients with RA and to compare serum protein profiles before and after ADA therapy. The study participants consisted of 20 patients with RA treated with ADA. Clinical periodontal and rheumatologic parameters and serum cytokine levels were evaluated at baseline and 3 months later. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant difference in abundance before and after ADA therapy were found and identified using mass spectrometry and protein databases. The patients showed a significant decrease in gingival index (P = 0.002), bleeding on probing (P = 0.003), probing depth (P = 0.002), disease activity score including 28 joints using C-reactive protein (P periodontal condition of patients with RA, which might be related to differences in serum protein profiles before and after ADA therapy.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis. PMID:21454471

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys(6), Lys(27), and Lys(29) linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys(6), Lys(27), and Lys(29) ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Nova onconeural antigens are neuron-specific RNA-binding proteins implicated in paraneoplastic opsoclonus-myoclonus-ataxia (POMA) syndrome. Nova harbors three K-homology (KH) motifs implicated in alternate splicing regulation of genes involved in inhibitory synaptic transmission. We report the crystal structure of the first two KH domains (KH1/2) of Nova-1 bound to an in vitro selected RNA hairpin, containing a UCAG-UCAC high-affinity binding site. Sequence-specific intermolecular contacts in the complex involve KH1 and the second UCAC repeat, with the RNA scaffold buttressed by interactions between repeats. Whereas the canonical RNA-binding surface of KH2 in the above complex engages in protein-protein interactions in the crystalline state, the individual KH2 domain can sequence-specifically target the UCAC RNA element in solution. The observed antiparallel alignment of KH1 and KH2 domains in the crystal structure of the complex generates a scaffold that could facilitate target pre-mRNA looping on Nova binding, thereby potentially explaining Nova's functional role in splicing regulation.

AIM: To investigate the prognostic significance of insulin-like growth factor-binding protein 3（IGFBP-3） in patients with cirrhosis.METHODS: Prospective study that included two cohorts: outpatients with stable cirrhosis（n = 138） and patients hospitalized for acute decompensation（n = 189）. Development of complications, mortality or liver transplantation was assessed by periodical phone calls and during outpatient visits. The cohort of stable cirrhosis also underwent clinical and laboratory evaluation yearly（2013 and 2014） in predefined study visits. In patients with stable cirrhosis, IGFBP-3 levels were measured at baseline（2012） and at second re-evaluation（2014）. In hospitalized subjects, IGFBP-3 levels were measured in serum samples collected in the first and in the third day after admission and stored at-80 ℃. IGFBP-3 levels were measured by immunochemiluminescence.RESULTS: IGFBP-3 levels were lower in hospitalized patients as compared to outpatients（0.94 mcg/mL vs 1.69 mcg/m L, P < 0.001） and increased after liver transplantation（3.81 mcg/m L vs 1.33 mcg/mL, P = 0.008）. During the follow-up of the stable cohort, 17 patients died and 11 received liver transplantation. Bivariate analysis showed that death or transplant was associated with lower IGFBP-3 levels（1.44 mcg/mL vs 1.74 mcg/m L, P = 0.027）. The Kaplan-Meier transplant-free survival probability was 88.6% in patients with IGFBP-3 ≥ 1.67 mcg/mL and 72.1% for those with IGFBP3 < 1.67 mcg/mL（P = 0.015）. In the hospitalized cohort, 30-d mortality was 24.3% and was independently associated with creatinine, INR, SpO2/FiO2 ratio and IGFBP-3 levels in the logistic regression. The 90-d transplant-free survival probability was 80.4% in patients with IGFBP-3 ≥ 0.86 mcg/mL and 56.1% for those with IGFBP3 < 0.86 mcg/mL（P < 0.001）. CONCLUSION: Lower IGFBP-3 levels were associated with worse outcomes in patients with cirrhosis, and might represent a promising prognostic

The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with β(1)-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages.

The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

This collaborative study characterizes a homogeneous standard for the protein content determination of granulocyte colony-stimulating factor (G-CSF) products with traceability of the measurement. The Kjeldahl method was used to determine the average protein content of G-CSF bulk as 2.505 mg/ml (95% C.I: 2.467-2.543 mg/ml, GCV 4.0%). Using G-CSF bulk as a traceability benchmark, the protein content of the final freeze-dried standard using reverse phase HPLC (RP-HPLC) was 215.4 μg protein per ampoule (95% C.I: 212.407-218.486 μg/ampoule, GCV 3.4%). A comparative study showed that there was no difference between using Filgrastim CRS (European Pharmacopeia G-CSF reference standard) and freeze-dried homogeneous standard when quantifying G-CSF protein content by RP-HPLC (P > 0.05). However, there were significant differences in the G-CSF protein content obtained using a serum albumin standard by Lowry assay and a G-CSF standard with RP-HPLC. Therefore, use of RP-HPLC with a freeze-dried homogeneous standard would eliminate the systematic errors introduced when using a serum albumin standard because of the differences in protein composition between the standard and the sample. It would also be helpful to use this method to compare the quality of G-CSF biosimilar products in situations where the protein content has been calibrated using various standards.

Introduction: Whether high factor (F)VIII and low free protein S levels are risk factors for arterial thrombosis is unclarified. Material and Methods: In a post-hoc analysis of a single-centre retrospective family cohort, we determined if these two proteins could increase the risk of arterial

Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compare to tissue. This revealed unexpectedly...... complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.......Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging due to highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art mass...

We have studied the effect of oxidative stress on protein synthesis in rat liver. Cumene hydroperoxide (CH) was used as an oxidant agent. The approach used was to determine the ribosomal state of aggregation and the time for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo. The results suggest that the elongation step is the most sensitive to CH treatment. The measurement of both carbonyl groups content and ADP-ribosylatable elongation factor 2 (EF-2), the main protein involved in the elongation step, indicates that under CH treatment EF-2 is oxidatively modified and a lower amount of active EF-2 is present. These results are corroborated by in vitro oxidation of EF-2 and could explain for the decline in the elongation step.

Full Text Available Solar ultraviolet (UV radiation is an important environmental factor that leads to immune suppression, inflammation, photoaging, and skin carcinogenesis. Here, we reviewed the specific signal transduction pathways and transcription factors involved in the cellular response to UV-irradiation. Increasing experimental data supporting a role for p38, MAPK, JNK, ERK1/2, and ATM kinases in the response network to UV exposure is discussed. We also reviewed the participation of NF-κB, AP-1, and NRF2 transcription factors in the control of gene expression after UV-irradiation. In addition, we discussed the promising chemotherapeutic intervention of transcription factors signaling by natural compounds. Finally, we focused on the review of data emerging from the use of DNA microarray technology to determine changes in global gene expression in keratinocytes and melanocytes in response to UV treatment. Efforts to obtain a comprehensive portrait of the transcriptional events regulating photodamage of intact human epidermis after UV exposure reveals the existence of novel factors participating in UV-induced cell death. Progress in understanding the multitude of mechanisms induced by UV-irradiation could lead to the potential use of protein kinases and novel proteins as specific targets for the prevention and control of skin cancer.

Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients. PMID:24781187

The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

Study of Drug-Protein Covalent Interactions by Mass Spectrometry A case study: Epidermal Growth Factor Receptor Abstract The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein belonging to the human EGFR family, which is in turn composed of four members: EGFR (ErbB1), ErbB2, ErbB3 and ErbB4. It is characterized by the presence of an extracellular ligand-binding domain, a transmembrane region, and a cytoplasmatic domain that is endowed with a tyrosine kinase (TK)...

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

Aerolysins are virulence factors belonging to the bacterial β-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells co-expressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the re-infection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.